The Ruling c

we have the sqi
nine squares, ea(
leukocyte counts
The very smalles
the triple ruled 1
never used for an
in a vaccine. It
make one of the

There are 400
large squares, th
4000 small squar

The unit in e
millimeter. The

In making a
mark 1 1 just abo

UNIVERSITY OF CALIFORNIA

MEDICAL CENTER LIBRARY

SAN FRANCISCO

FROM THE IIBRARY OF
ARTHUR P. KAELBER, M.D.

B first place,
made up of
tection with
rge squares.
:ersection of
;are and are
5 of bacteria
squares to

;re are nine
There are

3 the cubic

ich has the
suction we
solution of

fill the pipette to ,

glacial acetic acid in water is most satisfactory. This gives a dilution of 1-20.

Counting with the 2/3 inch objective all of the highly refractile dots representing
leukocytes in one of the i mm. squares at either of the four corners we note the num-
ber and mentally multiply by 20 (the number of times the blood was diluted). As
the depth of the diluted blood between the ruled surface of the haemacytometer
slide and the under surface of the cover-glass is only i/io of a millimeter, we multiply
the figure as above obtained by 10 to get the number of cells in a 1-20 dilution of
blood in a space of one cubic millimeter.

Example: Counted 90 leukocytes; 90X20 = 1800X10 = 18,000: equals number
of leukocytes in i cubic mm. of blood.

For red counts we use the red count pipette which has the 101 mark just above the
bulb. Taking up blood to 0.5 we draw up the diluting fluid to 101. This gives a
dilution of 1-200. Counting the red cells in five of the aggregations of 16 small
squares (1/20 mm.) thus having counted 80 small squares we have counted 1/50
of the total number of small squares in a cubic mm., there being 4000 small squares
in a cubic mm. Consequently the number of red cells in 80 small squares multiplied
by 50 and then by the dilution of 200 gives the number of red cells in one cubic
mm. of the blood examined.

It is well to make a second preparation and record the average of the two counts.

I

-//;

Jtrthur Protchf<t Xacller W?. S>.

7.5 yi

SvwedX

X. ^ ^

• •

•

Principal normal and pathological blood-cells with average size,
percentage in a normal differential count and the diseases in which
certain pathological cells are more or less pathognomonic.

The diameter of the bottom of this Petri dish is 3 inches or 7.5+
centimeters.

The area of a circle is equal to the square of the radius multiplied
by TT or 22/7.

i 1/2 in. = radius, i 1/2X1 1/2 = 2.25. 2.25X22/7 = 7.07 square
inches.

3.75 cm. = radius. 3.75X3.75 = 14-06. 14.06X22/7=44.1 square
centimeters.

Number of bacterial colonies in i sq. in. averages, approximate!]
75. Number in 7.07 sq. in. = 530.

Number of bacterial colonies in i sq. cm. averages, approximately,
12. Number in 44.1 sq. cm. = 528.

In*a microscopic field, if the diameter were 8 small squares (i/:
mm.), the radius would be 4 small squares and the area of such
round field would be 4X4=16X22/7 = 50+. Such a field woi
contain 50 small squares.

ire

:

BY THE SAME AUTHOR

The Diagnostics and Treatment

OP

Tropical Diseases

86 ILLUSTRATIONS 12110 433 PAGES
CLOTH, $2.00, POSTPAID

"We can thoroughly recommend Dr. Stitt's
work to every student and practitioner of tropical
medicine. It contains a large amount of sound
and up-to-date information presented in a concise
way in a comparatively small space." (From the
Lancet, London.)

"There are now so many books on tropical
medicine that any fresh addition to the ranks will
require to possess considerable merits to take a
place in the front rank, but this little work of
Stitt's will, we think, attain this position. Con-
sidering the space at the author's disposal it is
wonderfully full. Most of the subjects are well
and accurately treated, and are, in addition, well
illustrated." (From the British Medical Journal.)

P. BLAKISTON'S SON & CO.
PHILADELPHIA

PRACTICAL

Bacteriology, Blood Work

AND

Animal Parasitology

INCLUDING

Bacteriological Keys, Zoological Tables
and Explanatory Clinical Notes

BY

E. R. STITT, A. B., Ph. G., M. D.

MEDICAL DIRECTOR, U. S. NAVY; GRADUATE, LONDON SCHOOL OF TROPICAL MEDICINE; HEAD
OF DEPARTMENT OF TROPICAL MEDICINE, U. S. NAVAL MEDICAL SCHOOL; PROFESSOR OF
TROPICAL MEDICINE, GEORGETOWN UNIVERSITY; PROFESSOR OF TROPICAL MEDI-
CINE, GEORGE WASHINGTON UNIVERSITY; LECTURER IN TROPICAL MEDICINE,
JEFFERSON MEDICAL COLLEGE; MEMBER, NATIONAL BOARD OF MEDICAL
EXAMINERS; MEMBER, ADVISORY BOARD, HYGIENIC LABORATORY;
FORMERLY ASSOCIATE PROFESSOR OF MEDICAL ZOOLOGY,
UNIVERSITY OF THE PHILIPPINES

\ <

Fourth Edition, Revised and Enlarged
With 4 Plates and 115 Other Illustrations Containing 505 Figures

PHILADELPHIA

P. BLAKISTON'S SON & CO.

1012 WALNUT STREET

FIRST EDITION, COPYRIGHT, 1909, BY P. BLAKISTON'S SON & Co.
SECOND EDITION, COPYRIGHT, 1910, BY P. BLAKISTON'S SON & Co.

THIRD EDITION, COPYRIGHT, 1913, BY P. BLAKISTON'S SON & Co.
FOURTH EDITION, COPYRIGHT, 1916, BY P. BLAKISTON'S SON & Co.

TMK M A I* L K PKKMK YORK PA

PREFACE TO THE FOURTH EDITION

As noted in previous prefaces, this manual represents the notes of
a course along the laboratory side of internal medicine which has
seemed to the author practical and concise.

The exhaustion of a large edition of this laboratory guide in a little
more than two years would indicate a demand for a book of this char-
acter. By combining notes on points of clinical importance with
laboratory details such a presentation of the subject would appear of
more value to the student of internal medicine than the bare laboratory
technic. .

In view of the enormous advances in internal medicine since the
appearance of the last edition it has been very difficult to incorporate
new material and at the same time retain the pocket manual feature.
This has been done, however, by the greater use of paragraphs of
smaller type separating those printed in larger type, a plan which adds
to the accessibility of the contents as well as lessening the number of
pages. Notwithstanding such utilization of space it has been necessary
to increase the contents of the book by almost one hundred pages.

To further facilitate quick reference numerous additional bold type
headings of paragraphs have been introduced.

It has been a difficult matter to write concise discussions of such
subjects as acidosis, anaphylaxis, deficiencies of renal functioning, etc.,
where space was so limited.

Practically every chapter in the book has been carefully revised
and new material added to each. In some of the sections the changes
in our views during the past two or three years have been so marked
that it has been necessary to entirely rewrite large portions of such
chapters.

A new chapter, dealing with diseases of doubtful or only recently
determined etiology has been added to Part IV. In this will be found
discussions of the vitamine theory in beriberi and pellagra as well as
recent findings in connection with such diseases as typhus fever, Oroya
fever, verruga, rat bite fever, spotted fever ^pf the Rocky Mountains
and sprue.

fottea lever o
31413

VI PREFACE TO THE FOURTH EDITION

In the appendix a new section has been added on the chemical blood
examinations, and in that on insecticides the recent views as to the best
methods of destroying lice to control the spread of typhus fever and
relapsing fever have been incorporated.

There has also been added to the appendix a section dealing with
anatomical and physiological normals to furnish ready reference for
work in the pathological or chemical laboratory.

Among the tests more recently accepted as of practical value and
incorporated in this edition may be mentioned the following: Schick
test for diphtheria immunity, tests for recognition of acidosis, tests
for efficiency in renal functioning, PetrofTs method for culturing tuber-
cle bacilli, Wolff and Junghans' test for gastric carcinoma, Bronfen-
brenner's modification of Abderhalden's technic, tests as applied to
the duodenal fluid, Lange's colloidal gold test for general paresis,
Fontana's spirochete staining technic, Gluzinski's gastric carcinoma
test, and many others.

There have also been many new illustrations of animal parasites
substituted for those in the third edition which did not appear to have
sufficient teaching value.

E. R. S.

PREFACE TO THE THIRD EDITION

IN the preparation of the third edition of this laboratory manual it
soon became evident that the new material to be added would increase
the size of the book beyond that which would permit its being readily
carried in one's pocket. It has, however, been possible to keep the
size of the book within the limits considered desirable by the use of a
smaller type in a considerable proportion of the paragraphs so that in
this way and by increasing the number of lines on each page it has been
possible to add extensively to the subject matter and with only an in-
crease of about sixty-five pages.

The advantage attaching to more ready reference obtained by the
alternation of different sizes of type would appear to make this plan an
improvement over the old.

While the chapters dealing with bacteriology have been added to
and made to include more recent advances it will be noted that in the
section on animal parasitology the subject matter has been greatly
increased.

In the revision of the chapter on protozoa I am greatly indebted to
Professor Minchin's recent work on the Protozoa and in those relating
to arachnoids and insects to the very practical volume of Colonel
Alcock entitled "Entomology for Medical Officers."

The illustrations have" been added to and many which did not seem
to bring out sufficiently details of anatomy have been replaced by others
more satisfactory in that respect.

The three plates of the cestode, trematode and nematode ova were
drawn by Mr. L. Avery under the supervision of P. A. Surgeon Garrison,
U. S. N. and it is believed that they will be found more satisfactory
than similar plates contained in works on animal parasitology.

Several new tables have been added among which special attention
is called to the one on urinary findings in various diseases of the genito-
urinary system and also to the key to the intestinal bacteria attached
to the inside of the board cover.

A chapter on "Disinfectants and Insecticides" giving the practical
application of methods of carrying out these important Public Health
questions has been added.

vii

Vlii PREFACE TO THE THIRD EDITION

In the chapter on "Immunity" a modification of Emery's technic
for the Wassermann test has been incorporated — the use of Noguchi's
reagents with Emery's technic. The subject matter of the sections on
vaccines and anaphylaxis has been extensively revised.

E. R. S.

PREFACE TO THE SECOND EDITION

THE fact of the necessity for a second edition of this manual of lab-
oratory and clinical diagnosis in a little more than a year would indicate
that the original arrangement of material should be adhered to.

Each section of the book had been carefully revised and much new
matter added. In particular has that part of the book relating to ani-
mal parasitology been rewritten and almost doubled in extent, and a
chapter on Poisonous Snakes added. In the chapter on " Practical
Methods in Immunity" the most recent advances in the Wassermann
test and practical agglutination methods have been incorporated as well
as a brief discussion of the question of Anaphylaxis.

The section on " Clinical Bacteriology and Animal Parasitology of
the Various Body Fluids and Organs" has been revised to meet the most
recent advances in clinical diagnosis. This section not only answers
as a cross index to the importance of the various bacteria and animal
parasites in practical clinical work, but gives a concise, practical state-
ment as to how to proceed in the examination of various secretions and
excretions. This information is difficult to obtain in the larger works
on clinical diagnosis by reason of its being taken up under many differ-
ent headings.

A method is given for the making of differential counts in the same
preparation as that for making the leukocyte count which has the ad-
vantages of accuracy and the saving of time.

Several new illustrations have been added — the one of poisonous
snakes has been taken from Stejneger's report.

The plan of making this little volume a practical one has been con-
tinued in the second edition; theoretical considerations have been
brought out only when necessary to a proper understanding of some
recent or difficult laboratory method.

The very elementary considerations and definitions have not been
given because in order to present a compact and at the same time a
practical working guide it has been necessary to eliminate that which
seemed least essential. Furthermore, instruction in biological science
is now a part of the requirements of candidates for admission to the
various medical schools.

ix

X PREFACE TO THE SECOND EDITION

At the request of many who have found the book of assistance I
have added an outline of those methods in the chemical examination
of urine and gastric contents which have seemed to me to be most essen-
tial in the making of diagnoses. In the tropics I have found the deter-
minations of total nitrogen and nitrogen eliminated as ammonia to be
exceedingly valuable in diagnosis. Methods for such determinations,
as elaborated by Assistant Surgeon E. W. Brown, U. S. Navy, of the
U. S. Naval Medical School, have proven satisfactory and have been
incorporated in this section which is to be found in the Appendix.

Every effort has been made to keep the book within the limits of a
pocket manual.

Owing to my absence from the United States I have to thank Dr.
Charles S. Butler for correcting the proof. For the revision of the
index I am indebted to Mr. John P. Griest.

E. R. S.

PREFACE TO THE FIRST EDITION

WHILE a member of the Naval Examining Board and examiner in
bacteriology and clinical microscopy, I have during the past six years
had an opportunity to judge of the qualifications of several hundred
graduates of the various medical schools of the country from the stand-
point of practical application in the laboratory of that which they had
learned as undergraduates.

More particularly I have made it a point to ascertain from the suc-
cessful candidates, while under instruction at the Naval Medical School,
the features of their laboratory courses, which had seemed to them
most practical; such methods being subsequently tested in our own
class work.

As a result I have endeavored to incorporate in this manual methods
which have been submitted to the criticism of postgraduate students
from all the leading medical schools of the country, and which have been
considered by them adapted to the requirements of practical, speedy,
and satisfactory clinical laboratory diagnosis.

For the laboratory worker the most valuable asset is common sense
and he must be able to bring to mind the possibilities of the production
of various artefacts and results from trivial errors in technic. It has
been my object to point out where such mistakes may arise, the reasons
for obtaining results differing from those ordinarily obtained and the
means employed to eliminate as far as possible such results.

We are too apt to neglect the trivial details of stains, reaction of
media, and the like, yet it is only when every detail of technic has been
rigidly carried out that we are in a position to judge of the significance
of an object observed in a microscopical preparation.

In bacteriology, candidates were frequently able to give the cul-
tural and morphological characteristics of all the important pathogenic
organisms, yet when it was required of them to outline the procedure
by which they would differentiate members of the typhoid-colon groups
when encountered in a plate made from faeces, the problem appeared
to them impossible. They possessed the information, but did not know
how to apply it.

In practical work, organisms can only be separated culturally by the
use of Keys and for this reason Keys are given at the beginning of each

xi

xii PREFACE TO THE FIRST EDITION

division of bacteria. These enable one to quickly place the organism
isolated in its respective group. Only methods of differentiation which
are applicable in a physician's private laboratory are given. Practical
methods for making the final identification by agglutination or other
immunity tests are described. Technic for immunizing animals to
furnish such sera is given in detail.

The giving of the cultural characteristics in a systematic tabulated
Key gives space in the notes for presenting the salient points in the
pathological and epidemiological aspects of each organism.

I have endeavored to give a scientific yet practical classification of
the important pathogenic moulds, a subject about which there exists
greater confusion in the minds of students than for any other. In the
nomenclature I have followed Gedoelst's "Les Champignons Parasites."

In the chapter on Media Making, it is believed that anyone after
reading this section and following the instructions will be able to satis-
factorily and without the adjuncts of a large laboratory make any kind
of media. The directions as to titrations are given in detail because
it is beginning to be recognized that reaction of media in bacteriology
is of as great importance as staining is in blood work.

The section on Blood Work is practical and gives a method for mak-
ing a Romanowsky stain which is quick and reliable. The chapter on
Normal and Pathological Blood gives in a few pages the more important
points to be borne in mind in considering a possible diagnosis.

While there is no difference between the laboratory requirements of
medical work in the tropics and that in temperate climates, unless by
reason of such measures of diagnosis being indispensable in the tropics,
it has, however, been my endeavor to treat every tropical question,
whether in blood work, bacteriology, or animal parasitology, in a more
complete way than is usual in manuals of this character. Therefore it
is believed that this little book will be of great service to the laboratory
worker in the tropics.

It is only from working under Doctor Charles W. Stiles in his course
of laboratory instruction in Animal Parasitology in the United States
Naval Medical School that I feel justified in presenting a concise out-
line of the subjects in medical zoology which appear to me to be most
important for the physician.

The system of arranging tables, showing the families, genera, etc.,
in which each species belongs will, it is believed, greatly simplify the
matter of classification for the medical student. The points given

PREFACE TO THE FIRST EDITION xiii

under each parasite are believed to be practical ones. When a parasite
has only been reported for man two or three times, very little space is
given to it.

Part IV summarizes the various infections which may be found in
different organs or excretions of the body and embraces both bacterial
and animal parasites. Practical methods for examining material are
also given.

The chapter on Immunity, in which the theoretical side is immedi-
ately illustrated by the practical application will tend to simplify this
bug-bear of the medical student.

The illustrations have been selected with a view to bringing out
points which are difficult to state briefly in the text, and furthermore
they have been grouped together so that comparison of similar parasites
is possible without turning from page to page.

I have in particular to thank Hospital Steward Ebeling of the Navy
for his care in bringing out such details.

By reason of the authority of Braun, it has been considered sufficient
to give in the tables only the proper zoological name of the parasite as
given in the 1908 German edition. The synonyms have been omitted
for consideration of space.

The works chiefly consulted in addition to that of Braun have been :
Albutt's System of Medicine; Osier's System of Medicine; Muir and
Ritchie's Bacteriology; Mense's Tropenkrankheiten; Blanchard's Les
Moustiques; Guiart and Grimbert's Diagnostic; Ehrlich's Studies in
Immunity; Stephens and Christopher's Practical Study of Malaria;
Daniel's Laboratory Studies in Tropical Medicine; Manson's Tropical
Diseases; Gedoelst's Les Champignons Parasites; Neveu-Lemaire
Parasitologie Humaine; Chester's Determinative Bacteriology; Leh-
mann and Neumann's Bacteriology.

E. R. S.

CONTENTS

PART I. BACTERIOLOGY

CHAPTER I. — APPARATUS.

The Microscope, i — Apparatus for sterilization, 6 — Cleaning glass-
ware, 9 — Concave slides, fermentation tubes, n.

CHAPTER II. — CULTURE MEDIA.

Nutrient bouillon, 20 — Standardization of reaction, 21 — Sugar-free
bouillon, 23 — Glycerine bouillon, 25 — Peptone solution, 25 — Nutrient
agar, 26 — Glycerine agar egg medium, 27 — Gelatine, 28 — Litmus milk,
28 — Potato, 29 — Blood serum, 30 — Blood agar, 31 — Bile and faeces
media, 31-33 — Culture media for protozoa, 35.

CHAPTER III. — STAINING METHODS.

Loffler's methylene blue, 39 — Carbol fuchsin, 39 — Gram's method, 39
— Acid-fast staining, 41 — Neisser's stain, 42 — Capsule staining, 43 —
Flagella staining, 44 — Spore staining, 45 — Staining of protozoa, 45.

CHAPTER IV. — STUDY AND IDENTIFICATION OF BACTERIA. GENERAL CON-
SIDERATIONS.
Methods of isolating bacteria, 48 — Classification, 50 — Use of keys, 51.

CHAPTER V. — STUDY AND IDENTIFICATION OF BACTERIA. Cocci.

Key, 57 — Streptococci, 58 — Sarcinae, 62 — Staphylococci, 63 — Pneu-
mococcus, 64 — Gram-negative cocci, 66.

CHAPTER VI. — STUDY AND IDENTIFICATION OF BACTERIA. SPORE-BEARING

BACILLI.

Key, 73 — Anthrax, 75 — Cultivation of anaerobes, 78 — Malignant
cedema, 82 — B. botulinus, 82 — B. tetani, 84 — B. aerogenes capsulatus,

87-

CHAPTER VII. — STUDY AND IDENTIFICATION OF BACTERIA. BRANCHING,
CURVING BACILLI. MYCOBACTERIA. CORNYEBACTERIA.

Acid-fast bacilli, 91 — Tubercle bacillus, 92 — Leprosy bacillus, 97 —

Non-acid-fast branching bacilli, 101 — B. mallei, 101 — B. diphtherias,

102 — Hofmann's bacillus, 107 — B. xerosis, 108.
CHAPTER VIII. — STUDY AND IDENTIFICATION OF BACTERIA.

Gram-negative bacilli, Hemophilic bacteria, in — Influenza bacillus,

in — Priedlander's bacillus, 114 — Plague, 114 — Eberth, Gartner, and

Escherich groups, 118 — Typhoid, 119 — Dysentery, 125 — Chromogenic

bacilli, 129.
CHAPTER IX. — STUDY AND IDENTIFICATION OF BACTERIA.

Spirilla, 131 — Cholera, 131.

CHAPTER X. — STUDY AND IDENTIFICATION OF MOULDS, 136.
CHAPTER XI. — BACTERIOLOGY OF WATER, AIR, AND MILK.

Water, 149 — Milk, 155 — Air, 159.

xv

Xvi CONTENTS

CHAPTER XII. — PRACTICAL METHODS OF IMMUNITY.

Methods of obtaining immune sera, 165 — Agglutination tests, 168 —
Precipitins, 170 — Deviation of the Complement, 171 — Fixation of
the Complement, 172 — The Wassermann reaction, 174 — Opsonic
power and preparation of vaccines, 186 — Anaphylaxis, 192.

PART II. STUDY OF THE BLOOD

CHAPTER XIII. — MlCROMETRY AND BLOOD PREPARATIONS.

Micrometry, 197 — Haemoglobin estimation, 200 — Counting blood, 202
— Study of fresh blood, 206 — Blood films, 208 — Staining blood films,
211 — lodophilia, 214 — Occult blood, 216 — Acidosis, 218.
CHAPTER XIV. — NORMAL AND PATHOLOGICAL BLOOD.

Color index, 222 — Red cells, 222 — White cells, 224 — Eosinophilia, 231
— Leukocytosis, 232 — Lymphocytosis, 234 — Diseases with a normal
leukocyte count, 235 — The primary anaemias, 235 — Secondary anae-
mias, 237 — The leukemias, 238.

PART III. ANIMAL PARASITOLOGY

CHAPTER XV. — CLASSIFICATION AND METHODS, 243.

CHAPTER XVI.— THE PROTOZOA.

Rhizopoda, 251 — Flagellata, 259 — Infusoria, 273 — Sporozoa, 282—
The malarial parasite, 283.

CHAPTER XVII.— THE FLAT WORMS.

Flukes, 294 — Liver flukes, 297 — Intestinal flukes, 298 — Lung flukes,
299 — Blood flukes, 300 — Cestodes, 303 — Somatic taeniasis, 310.

CHAPTER XVIII.— THE ROUND WORMS.

Filariidae, 315 — Key to filarial larvae, 319 — Trichinosis, 321 — Hook-
worms, 325 — Ascaridae, 330 — Leeches, 332.

CHAPTER XIX. — THE ARACHNOIDEA.

The mites, 335 — The ticks, 338 — The Linguatulidae, 342.

CHAPTER XX.— THE INSECTS.

The Pediculidae, 345— The Diptera, 351— Biting flies, 352— Myiases,

359-
CHAPTER XXI.— THE MOSQUITOES.

Dissection of mosquitoes, 370 — Differentiation of Culicinae and Anoph-

elinae, 371 — Classification of Culicidae, 372.
CHAPTER XXII.— THE POISONOUS SNAKES, 377.

PART IV. CLINICAL BACTERIOLOGY AND ANIMAL

PARASITOLOGY OF THE VARIOUS BODY

FLUIDS AND ORGANS

CHAPTER XXIII.— DIAGNOSIS OF INFECTIONS OF THE OCULAR REGION, 381.
CHAPTER XXIV. — DIAGNOSIS OF INFECTIONS OF THE NASAL CAVITIES, 384.
CHAPTER XXV. — EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL,

386.
CHAPTER XXVI.— EXAMINATION OF SPUTUM, 389.

CONTENTS XV11

CHAPTER XXVII. — THE URINE, 393 — RENAL FUNCTIONING, 402.

CHAPTER XXVIII.— THE FAECES, 405.

CHAPTER XXIX.— BLOOD CULTURES AND BLOOD PARASITES, 412.

CHAPTER XXX. — THE STOMACH CONTENTS, 416 — DUODENAL FLUID, 417.

CHAPTER XXXI. — EXAMINATION OF Pus, 419.

CHAPTER XXXII. — SKIN INFECTIONS, 421.

CHAPTER XXXIII.— CYTODIAGNOSIS, 423— SPINAL FLUID, 425.

CHAPTER XXXIV. — RABIES, VACCINIA AND SMALLPOX, 429 — FILTERABLE

VIRUSES, 433.
CHAPTER XXXV.— DISEASES OF UNKNOWN ORIGIN, 435.

APPENDIX

PREPARATION OF TISSUES FOR EXAMINATION IN MICROSCOPIC SECTIONS, 443.

MOUNTING AND PRESERVATION OF ANIMAL PARASITES, 450.

PREPARATION OF NORMAL SOLUTIONS, 452.

CHEMICAL EXAMINATION OF BLOOD, 454.

CHEMICAL EXAMINATION OF THE URINE, 456.

CHEMICAL EXAMINATION OF THE GASTRIC CONTENTS, 469.

CHEMICAL TESTS OF DUODENAL FLUID, 472.

DISINFECTANTS AND INSECTICIDES, 472.

BACTERIOLOGY, BLOOD-WORK AND
ANIMAL PARASITOLOGY

CHAPTER I
APPAJRATUS

THE MICROSCOPE

THE most important piece of apparatus for the laboratory worker
is the microscope. Very satisfactory microscopes can be purchased in
this country. Instruments of standard German make are in use in
many laboratories and appear to give general satisfaction. It is impos-
sible to do good microscopical work unless the microscope gives and
continues to give good definition and the working parts remain firm.

Folding microscope stands are now made which are perfectly satisfactory, such
instruments, however, have only the advantage of occupying less space in a case so
that unless the question of compactness is involved, as in an outfit for the military
services or for a microscopist who travels about a great deal, the ordinary rigid horse-
shoe base is to be preferred.

A mechanical stage is almost a necessity in connection with blood-work and its use
is advantageous in bacterial preparations. For the study of tissue sections the mov-
ing of the slide with the fingers is preferable. Therefore, the mechanical stage should
be capable of ready attachment or removal. For the examination of colonies
growing in Petri dishes we also use the stage unencumbered with the mechanical
stage. A triple or quadruple nose-piece, according to the number of objectives used,
is also indispensable.

One should always use a magnifying glass or better a lens in a tripod or an apla-
natic^triplet in the study of microscopic objects, prior to examination with the mi-
croscope. A dissecting microscope is better for this purpose and is very useful in
dissecting mosquitoes, etc. In fact the dissecting microscope is almost essential in
the examination of helminthological and entomological specimens. Of particular
value is a stage forceps for orienting insects, especially mosquitoes, when examined
on the stage of either a compound or simple microscope.

The following precautions should be observed to prevent injury to
the microscope:

i. If the fine adjustment works through the arm of the microscope, always grasp
the instrument by the pillar which supports the stage. In those microscopes, how-

2 APPARATUS

ever, which are not constructed in this way the arm is made to serve in lifting the
instrument.

2. Always keep the microscope in its case or covered with a bell jar when not in
use in order to keep away the dust. A piece of black cloth supported on a wire
lamp-shade frame makes a most convenient protecting covering.

3. Alcohol ruins the lacquer of an instrument and care should be observed to keep
all parts of the microscope from coming in contact with acids, alkalies, chloroform or
xylol as well as alcohol.

4. Always use Japanese lens paper in wiping off the dust from dry objectives or
the immersion oil from the 2-mm. one. Should one neglect to wipe off the oil from
the oil-immersion objective, the dried oil can be removed by wiping with a drop of
xylol on lens paper, but the cleaning should be done as rapidly as possible, with a
final wiping off with dry lens paper, to avoid damage by the xylol to the setting of
the lenses. Throw lens paper away after using it once.

5. Lenses are very liable to deteriorate in the tropics. One should be careful to
protect his instrument from the direct light of the tropical sun.

6. If any oil is used on the mechanical parts for lubrication, all excess should be
wiped off to avoid the catching of dust or gritty particles.

Objectives. — To meet the demands of clinical microscopy there
should be three objectives, preferably a i6-mm. (%-inch), a 4-mm.
(%-inch) and a 2-mm. (J^-indi) homogeneous oil immersion one.

The Zeiss A A is a ly-mm. objective, and the Leitz No. 3, an i8-mm. or ^-inch
one. The Zeiss D is about 4. 2-mm. and the Leitz No. 6, 4.4-mm. or ^-inch. A
dustproof quadruple nose-piece with four objectives will be found a great convenience
(in addition to the %-inch and ^2-mcn objectives, a ^-inch for urine and blood
counting, with a ^-inch for examining hanging-drop preparations and for quick
examination of blood smears). An apochromatic objective costs about three times
as much as an achromatic one and, except in photographic work, has little if any
advantage.

Oculars. — As regards oculars (eye-pieces) a Leitz No. i and a No. 4
will best meet the requirements. For high magnification a No. 8 may
be of service. The Zeiss oculars are numbered according to the amount
they increase the magnification given by the objective; thus a No. 2
increases the magnification, given by the objective alone, twice; a No.
8, eight times.

Some oculars are classified according to their equivalent focal distance and are
referred to as H-inch, i-inch and 2 -inch oculars.

A i-inch or 25-mm. ocular magnifies the magnification produced by the objective
about 10 times while a 2-inch or 5o-mm. one increases the magnification of the ob-
jective four times.

A Leitz No. o is a 5o-mm. ocular and magnifies four times. The Nos. i, 2, 3, 4
and 5 are 40, 35, 25, and 20 mm. respectively and give eye-piece magnifications of 5,
6, 8, 10 and 12 times.

The oculars in common use are known as negative or Huyghenian oculars, by

OBJECTIVES 3

which is meant an ocular in which the lower lens (collective) assists in forming the real
inverted image which is focused at the level of the diaphragm within the ocular.
When using a disc micrometer, it is supported by this diaphragm, and the outlines of
the image are cut by the rulings on the glass disc, and so we are enabled to measure
the size of the object examined. The measurement of various bacteria, blood-cells,
and parasites is exceedingly simple and assists greatly in the study of bacteria, and is
indispensable in work in animal parasitology. (For details of micrometry see section
on blood- work.) When an ocular is termed positive, it refers to an ocular which acts
as a simple microscope in magnifying the image, the image being formed entirely by
the objective and located below the ocular. By fixing one end of a hair on the rim of
the diaphragm inside the ocular with a minute drop of balsam one has a satisfactory
pointer to locate any particular cell in the microscopic field.

Objectives are usually designated by their equivalent focal distance. It is impor-
tant to remember that the equivalent focal distance does not represent the working
distance of an objective, by which is meant the distance from the upper surface of
the cover-glass to the lower surface of the objective. Thus a K-inch objective may
have to be approached to the object so that the distance intervening may be only
% inch or even less. This explains the frequent inability to focus an object when a
high-power dry objective (^-inch or ^-inch) is used with a rather thick cover-glass
— the objective possibly having a short working distance, so that the thickness of
the cover-glass does not allow of any free working distance.

Instrument makers generally specify the thickness of cover-glass to be used with
a certain tube length, but as a practical matter it will be found convenient to use No .
i (very thin) cover-glasses. The principal objection to these is that they are more
fragile than the No. 2, but with a little practice in cleaning cover-glasses this is
negligible. Immersion lenses are less affected than dry lenses by the question of a
certain thickness of cover-glasses for a certain tube length.

One of the most fruitful causes of the crushing of microscopical ob-
jects and the overlying cover-glass or, what is far more important, the
breaking of the cover-glass of a hanging-drop preparation and conse-
quent risk of infection is the attempt to focus with the fine adjustment.

It should be an invariable rule for the worker to bring his objective practically into
contact with the upper surface of the cover-glass, then using the coarse adjustment
(rack and pinion) to slowly elevate the objective, looking through the eye-piece at
the same time. In other words, obtain focus with the coarse adjustment and main-
tain it with the fine adjustment (micrometer screw). The fine adjustment should
only be used after the focus is obtained.

Oil Immersion. — In using the oil-immersion objective always dip the
lens in the oil and practically touch the cover-glass — the eye being at
a level with the stage — before beginning to focus. With the coarse ad-
justment one can feel the contact with the cover-glass, which is impos-
sible with the fine adjustment. It saves time and disappointment to
make a preliminary examination of a preparation requiring the high

4 APPARATUS

dry or immersion lens with a low power (%-inch) before employing the
higher power; in this way we locate or center a suitable field for study.

It will be observed that objectives frequently have their numerical aperture
marked on them. This is expressed by the letters N.A. From a practical stand-
point this gives the relative proportion of the rays which proceeding from an object
can enter the lens of the objective and form the image. Of course, the greater the
number of rays, the greater the N.A., the better the definition, and consequently the
better the objective. Immersion oil, having the same index of refraction (1.52)
as glass, would not deflect rays coming from the object and so prevent their enter-
ing the objective, as would be the case if we used a dry objective with an interven-
ing air space. In this case a portion of the rays would be turned aside by the differ-
ence in the refractive index of air. As a rule, the higher the numerical aperture,
the better the objective and the less the working distance. In blood counting, the
cover-glass being comparatively thick, it may happen that with a ^j-inch of high
numerical aperture there may not be sufficient working distance to bring the blood-
cells into focus, which could be done with an objective of lower numerical aperture.
Consequently, we must always consider the matter of working distance as well as
that of numerical aperture. The skill of the optician, however, can obviate this
defect in an objective of high numerical aperture so that it may combine the qualities
of perfect definition with sufficient working distance.

Practical Points in the Use of the Microscope. — An important matter
in the use of the microscope is to get all the details possible with a low
power before using a higher power. This, of course, does not apply to a
bacterial preparation where it is necessary to use a J^2~mch or a high-
power dry lens.

It is well, however, in a bacterial or blood preparation to first examine the smear
with the %-inch objective in order to determine suitable areas for examination with
the oil-immersion objective. With tissue sections it is not only advisable to begin the
study with the lowest power, but even an examination with the unaided eye or with
a magnifying glass, before using the microscope, will give a surprising amount of
information.

After using the oil-immersion objective the lens should be wiped clean of oil
with a strip of Japanese lens paper or with a silk handkerchief. If the oil should
dry on the surface of the lens it may be removed with a drop of xylol on a piece of
lens paper. Immediately afterward the lens should be dried. Dried oil on a lens
often causes the lens to be considered defective. Accidental contact of the dry
objectives with oil is not uncommon and should always be thought of when satis-
factory optical effects are not obtainable. In depositing the drop of immersion oil
on the slide bubbles are at times formed which make it almost impossible to use the
H 2-inch objective. Under such circumstances I either prick the bubbles or wipe off
the oil and deposit a drop anew.

It is advisable to cultivate the use of both eyes in doing microscopical work.
When using one eye the other should be kept open with accommodation relaxed.
It is this squinting of the unemployed eye which so often fatigues. A strip of card-

ILLUMINATION 5

board 4 or 5 inches long, with an opening to fit over the tube of the microscope,
leaving the other end to block the vision of the unused eye, will prevent the strain.
This apparatus can be purchased in vulcanite.

Warm Stages. — A warm stage for the study of living protozoa may be extempor-
ized by taking a piece of copper about the size of the stage and with a strip projecting
out anteriorly for 5 or 6 inches. The under surface of the plate is covered with
flannel and a hole about i inch in diameter cut out of the center. The proper amount
of heat is applied by a flame impinging on the tongue-like projection of the copper
plate.

At present there are electrically heated warm stages, connected with the desk
socket by a wire and plug, which are most convenient. In fact they are almost as
satisfactory as the more expensive and less convenient warm chamber or microscope
oven surrounding the microscope.

Illumination. — Direct sunlight or excessively bright light is to be
avoided. If such conditions must exist a white shade or muslin curtain
drawn across the window is a necessity. Light from the north and from
a white cloud is the most desirable. South of the equator a southern
light. In the tropics a piece of plate glass fitted into the lower part of
a wire screen frame gives good lighting, keeps out dust, and does not
interfere greatly with the circulation of the air.

The technic in connection with proper illumination is probably more important
than any other point; unless the light is utilized to the best advantage, the best
results cannot be obtained. In examining fresh blood preparations or hanging drops
the concave mirror should be used and the light almost shut off by the iris dia-
phragm so as to give a contour picture. In examining a stained blood or bacterial
preparation, the Abbe condenser should be properly focused so as to best illuminate
the stained film. In many instruments set-screws are provided which check the ele-
vation of the Abbe condenser when the proper focus is reached. Inasmuch as the
light from the condenser should come to a focus exactly level with the object studied,
it is evident that a fixed position for the condenser would not answer when slides of
different thickness were used. Always use the plane mirror when examining stained
bacterial or blood films, as a color image is desired. Ordinarily in examining tissue
sections, the Abbe condenser should either be put out of focus by racking down or by
the use of the concave mirror and the narrowing of the aperture of the iris diaphragm.
Swing-out condensers are now made which are very convenient. The proper em-
ployment of illumination only comes with experience, and one should continue to
manipulate his mirrors, diaphragm, and condenser until the best result is obtained.
Then study the specimen.

For microscopical work in a laboratory not properly supplied with windows or for
night work the frosted incandescent bulb is very satisfactory.

An objection to artificial light is that one working almost entirely
with sunlight forms standards and when using a different light is some-
what confused in interpretation of the microscopical picture.

6 APPARATUS

There is now on the market a special electric lamp and light filter which tends to
give optical effects similar to that obtained with sunlight. These are called daylight
filters. The bulb of the lamp is filled with nitrogen gas and the glass filter screen has
a bluish color. By using a 6-inch round-bottom flask filled with an alkaline solution
of copper sulphate as a condenser, similar illuminating effect to that of the day-
light lamp can be obtained.

Dark Ground Illumination. — Very valuable information, especially
as regards the detection of treponemata in material from hard chancres
or mucous patches, may be obtained by the use of dark ground illumina-
tion. There are many different types of apparatus for this purpose.

The bacteria or spirochaetes are intensely illuminated and show as
brilliant silvery objects in contrast to the dark background.

When the morphological details of a brightly illuminated object in the dark field
can be distinctly observed it is proper to use the term dark ground illumination.
When only particles, usually surrounded by bright and dark rings, and not showing
any structure, are observed in the dark field the proper designation is ultramicro-
scopic. An apparatus using only the short waves of the ultra-violet spectrum
enables one to observe particles no larger than Ho of a micron. For this apparatus
it is necessary to employ photographic plates. In using the H 2-inch objective with
dark ground illumination a funnel-like base is supplied on which we screw the nickle-
plated front mount of the objective. Before using the dark-field apparatus it must
be centered with a low power. This is carried out by getting concentric rings paral-
lel with the circle of the microscopic field. Immersion contact between the front
surface of the Abbe condenser and the under surface of the slide carrying the prepa-
ration must be made before focussing the ^2th objective. As a source of illu-
mination we may use a small arc-lamp or a Nernst lamp or an incandescent gas lamp.
In using an arc-lamp one must have a suitable rheostat according to the electrical
current employed. Information as to voltage and nature of current must be given
the one supplying the apparatus.

In making preparations the slides and cover-slips should be scrupulously clean
and the material thinly spread out and free of bubbles. With low power objectives
one can obtain satisfactory dark-field illumination by pasting a circle of.black paper
in the center of one of the glass discs which fit in the ring under the lens of the sub-
stage condenser. The diameter of the opaque center will have to be greater as the
magnifying power of the objective increases and for oil-immersion objectives a special
apparatus is required. Flagellates in faeces are best studied with the dark-field
illumination.

.APPARATUS FOR STERILIZATION

For the purpose of sterilizing glassware, media, and old cultures there
are three methods ordinarily employed. Th« hot-air sterilizer, in which
a temperature of about i5o°C. is maintained for one hour, is ordinarily
used for the sterilization of Petri dishes, test-tubes, pipettes, etc.

STERILIZERS 7

If the temperature is allowed to go too high, there is danger of charring the cotton
plugs and also of causing the development of an empyreumatic oil which makes the
plugs unsightly and causes them to stick to the glass. Again we must be careful not
to open the door until the temperature has fallen to 6o°C., otherwise there is danger
of cracking the glassware. Where gas is not obtainable, the hot-air sterilizer is not a
very satisfactory apparatus.

FIG. i.— Dressing sterilizer showing cylinder containing water (K) heated either
by gas or Primus kerosene lamps.

Arnold Sterilizer.— The Arnold sterilizer is to be found everywhere
and can be used on blue-flame kerosene-oil stoves as readily as with gas
burners. The most convenient form, but more expensive, is the Boston
Board of Health pattern. The ordinary pattern, with a telescoping
outer portion, answers all purposes, however.

8 APPARATUS

In the Arnold, sterilization is effected by streaming steam at ioo°C. It is usual to
maintain this temperature for fifteen to twenty-five minutes each day for three suc-
cessive days. The success of this procedure — fractional sterilization — is due to the
fact that many spores which were not killed at the first steaming have developed into
vegetative forms within twenty-four hours, and when the steam is then applied such
forms are destroyed. Experience has shown that all the spores have developed by
the time of the third steaming, so that with this final application of heat we secure
perfect sterilization.

It is customary to use the Arnold for sterilizing gelatin and milk
media, even when the autoclave is at hand, the idea being that the
greater heat of the autoclave may interfere with the quality of such
media. The most convenient autoclave is the horizontal type, such as
is to be found everywhere for the sterilization of surgical dressings.

The source of heat may be either electricity, gas, the Primus kerosene-oil lamp or
steam from an adjacent boiler. More recently a method of employing kerosene, gaso-
lene, or alcohol with a gravity system has been perfected. During the past ten years,
in the laboratory of the U. S. Naval Medical School, we have been using a dressing
sterilizer, made by the American Sterilizer Co., with which it has been possible to
most satisfactorily carry out all kinds of sterilization, thus doing away with the use
of the Arnold and the hot-air sterilizer. It is impossible to sterilize ordinary fermen-
tation tubes in the autoclave on account of the boiling up of the media and wetting
of the plugs. This is still done with the Arnold. By use of the Durham tubes —
which are to be preferred, except for gas analysis — sugar media can be thus sterilized.

Should a small bubble remain in the top of the small inverted inner tube after
removal from the autoclave, one may make a mark with a grease pencil at the line
of the bubble; or, if preferred, the basket of Durham tubes can be heated to boiling
for ten minutes in a pan of water or in the Arnold when, after cooling, the bubble will
be found to have disappeared.

Glassware will come out from such an autoclave with wrappers as dry and plugs
of the test-tubes as stopper-like as could be effected in a hot-air sterilizer.

The objection which exists in the use of some autoclaves, as regards condensa-
tion on dressings or apparatus, does not exist in this type. The mechanism, by
which the inner and outer chambers are connected and disconnected, and that for
vacuum production, rests in the simple turning of a lever from mark to mark. We
have been able with a gas burner to obtain a pressure of 15 pounds in less than ten
minutes. In sterilizing test-tubes we place them in small rectangular wire baskets,
6X5X4 inches. These baskets are to be preferred to round ones, as they pack more
satisfactorily in the refrigerator used for storing media. In sterilizing flasks, test-
tubes, Petri dishes, throat swabs, pipettes, etc., it has been our custom, after ex-
posing to 20 pounds' pressure for twenty minutes, to produce a vacuum for two or
three minutes; then with the steam in the outer jacket for a few minutes to thor-
oughly dry the articles in the disinfecting chamber. The valve to the inner chamber
is then opened to break the vacuum; the door is now opened, and the articles re-
moved in as dry a state as if they had been in the hot-air sterilizer. Articles, how-
ever, can be thoroughly dried without the use of a vacuum, simply allowing the steam
to remain in the outer jacket with the steam cut off from the inner chamber.

CLEANING GLASSWARE 9

PRESSURE AND TEMPERATURE TABLE

5 pounds' pressure, 107. 7°C., 226°F.

10 pounds' pressure, 115. 5°C., 24o°F.

15 pounds' pressure, 121. 6°C., 25o°F.

20 pounds' pressure, i26.6°C., 26o°F.

25 pounds' pressure, 130. 5°C., 267°F.

30 pounds' pressure, 134. 4°C., 274°F.

All such articles as Petri dishes, pipettes, swabs, etc., are wrapped in cheap
quality filter-paper, making a fold and turning in the ends as is done in a druggist's
package. Old newspapers answer well for this purpose. The sterile swab can be
used for many purposes in the laboratory. They are most easily made by taking
a piece of copper wire about 8 inches long, flattening one end with a stroke of a
hammer, then twisting a small pledget of plain absorbent cotton around the flat-
tened end. After wrapping, the swabs are sterilized in bunches. We not only use
them for getting throat cultures, but in addition for culturing faeces, pus, or other
such material. The pus is obtained with a swab, which material is then distributed
in a tube of sterile bouillon or water. With the same swab the surface of an agar
plate is successively stroked. This method is almost as satisfactory as the German
one of using bent glass rods for this purpose. Everyone has encountered the diffi-
culties attendant upon the bending of platinum wires and also the possibility of
destroying your organisms by an insufficiently cooled wire.

CLEANING GLASSWARE

It is a routine in our laboratory for everything to go through the
sterilizer at i25°C. before anything else is done. This is a safe rule
when dealing with dangerous pathogenic organisms (especially tetanus
and anthrax).

As soon as taken out of the sterilizer the contents are emptied, and the tube or
dishes placed in a i % solution of washing soda and boiled. This thoroughly cleans
them. As the washing soda slightly raises the boiling-point and also makes the
spores more penetrable, it would appear that under ordinary circumstances, it would
be sufficient to place all contaminated articles in a dishpan with the soda solution,
and boil for at least one hour, not using a preliminary sterilization in the autoclave.
The tubes are now cleaned with a test-tube brush, thoroughly rinsed with tap water
and placed in a i% solution of hydrochloric acid for a few minutes; then rinsed
thoroughly in water and placed in test-tube baskets, mouth downward, and allowed
to drain over night. Some laboratory workers boil their test-tubes and other glass-
ware in water containing soap or soap powder and, after a thorough rinsing in tap
water, drain. Hydrochloric acid should not be used after the soap as it will cause
the formation of an unsightly coating difficult to remove. When thoroughly dry
they may be plugged and sterilized. To plug a test-tube, pick out a little pledget of
plain absorbent cotton about 2 inches in diameter from a roll. Place it over the cen-
ter of the tube and with a glass rod push the cotton down the tube about an inch. In

IO

APPARATUS

culturing slow growing organisms, as tubercle bacilli, or certain pathogenic protozoa
it is necessary to have plugs so prepared as to prevent drying out of the medium in the
tube. The simplest way of accomplishing this is to melt some paraffin in a pan, then
removing the cotton plug with the fingers to dip the end entering the tube into the
melted paraffin and then push the surface so prepared into the tube. Plasticine,
sealing wax or paraffin may be used to seal over the tops of such test-tubes.

Cleaning Fluid. — The cleaning fluid commonly used in laboratories consists of i
part each of potassium bichromate and commercial sulphuric acid with 10 parts of
water. This is an excellent mixture for cleaning old slides, etc., especially when
grease or balsam is to be gotten rid of. It is very corrosive, however.

FIG. 2. — i, Inoculation of tubes; 2, plugging of tubes; 3, filling tubes; 4, Smith's
fermentation tube; 5, Durham's fermentation tube.

In cleaning glassware for such tests as the colloidal-gold one it is essential that we
use such a cleaning fluid. An efficient and less corrosive method for cleansing slides
and cover-glasses is to leave them over night in an acetic acid alcohol mixture (two
parts of glacial acetic acid to 100 parts of alcohol). After drying and polishing out
of this mixture, it is well to pass the slides and cover-glasses through the flame of
a Bunsen burner or alcohol lamp to remove every vestige of grease. Ordinarily,
rubbing between the thumb and forefinger with soap and water, then drying with
an old piece of linen, and finally flaming will yield a perfect surface for making a
bacterial preparation.

HANGING DROP II

CONCAVE SLIDES, FERMENTATION TUBES

The concave slide is ordinarily used for making hanging-drop prepa-
rations for the examination of bacteria as to motility, capsules, size
and arrangement.

To prepare a hanging-drop preparation for the study of motility it is best to place
a loopful of the young bouillon culture or a loopful of salt solution into which is then
emulsified a small amount of growth from an agar slant, in the center of the cover-
glass; now having applied with a brush a ring of vaseline around the concave depres-
sion in the slide we apply the slide as a cover to the cover-glass which latter adheres
to the ring of vaseline. The completed hanging-drop preparation can now be turned
over and placed on the stage of the microscope.

A substitute which is equally good may be made by spreading a ring or square of
vaseline — smaller than the cover-glass to be used — in the middle of a plain slide.
Then putting a loopful of salt solution in the center of the space, and inoculating with
the culture to be studied, we finally cover it with a cover-glass, gently pressing the
margins down on the vaseline. This gives a preparation for the study of motility or
agglutination which does not dry out for hours, and is easier to focus upon than the
concave slide hanging-drop preparation.

Hanging Drop. — In examining a hanging drop first use a low-
power objective and, having brought into focus the margin of the drop

FIG. 3. — Hanging drop, over hollow ground slide. (Mac Neal.) »

as a center line, change to a J£- or J^-inch objective. By this pro-
cedure a thin layer of fluid is brought under the high dry objective
instead of the deeper layer in the center of the drop. It is not ad-
visable to use an immersion objective with a hanging-drop preparation.

The light should be cut down to a minimum with the iris diaphragm and the con-
cave mirror used. When we have finished examining the preparation the cover-glass
should be pushed over with the forceps so that a corner projects and we then seize
this with the forceps, lift up the cover-glass and drop it into the disinfecting solu-
tion along with the slide.

Fermentation Tubes. — The fermentation tube with a bulb and closed
arm is expensive, difficult to clean, and is easily broken. It is, however,
convenient in the determination of the gas formula of an organism.
Its use is described under water analysis. As a substitute in the study
of gas production and in water bacteriology, the Durham tube is to be
recommended.

12

APPARATUS

Into a test-tube, about 1X7 inches, we introduce the special sugar media, then
drop down a -small test-tube (KX3 inches) with its open end downward. Insert
the plug of the large tube and sterilize. During sterilization the fluid enters the
mouth of the smaller tube and fills it, and when the medium is subsequently inocu-
lated, if gas forms, it appears in the upper part of the closed end of the smaller tube.

FIG. 4. — Blood serum coagulating apparatus.

Inspissators. — For inspissating blood-serum slants a regular inspissa-
tor is desirable.

This is nothing more than a double-walled vessel, the space between
the walls being filled with water.

As a^ substitute one may take the common rice cooker (double boiler). Fill the
outer part with water; and in the inner compartment pack the serum tubes properly

slanted on a piece of wood or a wedge-shaped
layer of cotton. Place a weight on the cover
of the inner compartment to sink it into the
surrounding water, and allow to boil for one
or two hours. This same apparatus may be
used for their sterilization on two subsequent
days, but it is better to sterilize in the auto-
clave or Arnold. The rice cooker is of the
greatest use in preparing culture media. For
this purpose the outer compartment is filled
with calcium chloride solution or a 25% solu-
tion of common salt, so that the tempera-
ture of the contents of the inner receptacle may be raised to the boiling point.
Of course media may be prepared in an ordinary sauce-pan but there is great
danger of scorching media prepared in this way. Special vessels with two bottoms,
between which is an air space, are now on the market and have an advantage
over the double boiler (rice cooker).

Ebony Finish. — As regards a working desk, it will be found convenient to have an
arrangement similar to the ordinary flat-top desk, with a tier of drawers on each side.

FIG. 5.— Rice cooker.

EBONY FINISH 13

A block of wood with holes bored in it to contain dropping-bottles may be placed in
the upper left-hand drawer. In this way the stains are as accessible as if they encum-
bered the desk. It is advisable to paint the inside of this drawer black so that the
light may not cause the staining reagents to deteriorate.

A very popular method of preparing the wooden surfaces of labora-
tory desks, sinks, and tables is the application of the so-called "acid-
proofing." This gives an ebony-like finish which is not affected by
strong acids.

In using it the surface of the wood must be new (free of any varnish, oil, or paint,
if previously so coated the surface must be planed).

Solution i

Potassium chlorate 125 .o grams.

Cupric sulphate 125 . o grams.

Water 1000 . o c.c.

Apply two coats of this solution at least twelve hours between applications.
When thoroughly dry apply two coats of solution No. 2.

Solution 2

Aniline oil 120.0 c.c.

Hydrochloric acid 180.0 c.c.

Water 1000.0 c.c.

When the treated surface is thoroughly dry apply one coat of raw linseed oil with a
cloth. After this is dry wash with very hot soapsuds.

An aspirating bottle on a shelf elevated 2 feet, with rubber tubing and glass
tip leading to a small aquarium jar or other desk receptacle, makes a good substitute
for a small sink and faucet. A Hofmann screw clamp on the rubber tube controls
the flow of water.

Ordinary glass salt cellars will be found very useful, where the watch-glass is
employed. They may also be wrapped, sterilized, and used to contain fluids for
inoculating, etc.

A glass-topped fruit jar or a specimen jar containing a disinfecting solution for con-
taminated slides, etc., should be on every working desk. A good solution is that of
Harrington (corrosive sublimate, -0.8; commercial HC1, 60.0 c.c.; alcohol, 400.0
c.c.; water, to 1000.0 c.c.).

Disinfectant Solution. — A very simple method of making a disinfec-
tant similar to lysol is to put one part of cresol or crude carbolic acid
and one part of soft soap in a wide-mouthed bottle over night. The
resulting compound (Liquor cresolis comp. U. S. P.) makes a perfect
solution with water and a 5% solution of this will be found at least
equal jto a 5% phenol solution. In addition to using as a desk jar dis-
infectant it is excellent for disinfecting faeces, sputum, etc.

14 APPARATUS

Platinum Loops.— For use in making loops and needles, platinum wire of 26 gauge
will be found most suitable. The handle made of glass rod is preferable to the
metal ones. One end is fused in the flame and, holding the 3- to 4-in. piece of plati-
num wire, with forceps, in the same flame, insert the glowing metal into the molten
glass. By taking two lengths of platinum wire and twisting them together a more
rigid needle is made for inoculating stab cultures.

Isolation cf colonies uslny two halves cf
ayar plate Instead cf two separate plates.

Zlnsser's anaerobic
plate, method

gutter's vaccine
ampule filler

FIG. 6. — i, Method of using one plate instead of two for isolation of colonies,
(see page 50). The separated colonies on the No. II side of the plate are studied
with unaided eye and aplanatic triplet (^ in. or % in.) both by reflected and trans-
mitted light. After we have determined the presence of two or more different
kinds of colonies, a well separated one of each type is selected and a blue pencil ring
made around it on the glass surface of the back of the Petri dish together with a num-
ber. This number is carried along on culture tubes or microscopical slide prepara-
tions until the organism is identified. 2, Lyon tube for blood. More convenient
than the Wright tube. For description see page 18. 3, Bronfenbrenner's an-
aerobic tube. This is made by drawing out a single test-tube instead of using two
separate tubes as with the Noguchi method. Before drawing out the test-tube the

Eiece of sterile tissue is introduced and after drawing out in the flame the ascitic
roth or sheep serum water, as well as the material for culturing, is introduced
through the drawn-out neck with a bulb capillary pipette. The lower part of the
tube is placed in water at 37°C. and the vacuum connection made and after exhaus-
tion of air the sterile paraffin oil is run in. The drawn-out portion is then sealed off
in a small flame and looks like a sealed vaccine ampule. 4, Noguchi apparatus.
5, Zinsser method for anaerobic plates (page 80). 6, Butler's apparatus for filling
vaccine ampules. The sterile vaccine is put in the sterile flask, and the stopper with
air intake and needle filler separately sterilized and then introduced into neck of
flask which is then inverted.

A platinum loop made around a piece of copper wire, 4 mm. in diameter holds
about 2 mg. of culture taken up from an agar slant. Kolle estimates that an
agar slant of typhoid bacilli or of staphylococci should contain 15 such loopfuls

INCUBATORS 1 5

while a streptococcus slant would have almost five. It has been estimated that such
a standard loop would contain between 2,000,000,000 and 3,000,000,000 organisms.
Of the greatest use in culturing material obtained at autopsy is the platinum spud.
This can be made by hammering out one end of a piece of 15- to i8-gauge platinum
wire.

For making smears from faeces, sputum, and the like, wooden tooth-
picks are very convenient; the kind with the spatulate end is preferable.

Incubators. — When gas is obtainable, the maintaining of a constant temperature
for the body temperature incubator (38°C.) and the paraffin oven (6o°C.) is best
secured by the use of some of the various types of thermo-regulators. The Reichert
type is the one in general use, although there are many features about the Dunham
and Roux regulators which are advantageous. •

If the pressure of the gas-supply varies from time to time, it is essential to regulate
this by the use of a gas-pressure regulator (MurrilPs is a cheap and satisfactory
one).

Incubators, controlled electrically, can be obtained of certain foreign makers, and
are quoted in catalogues of American dealers. It is probable that the Koch petro-
leum lamp incubator is the most satisfactory one where gas is not obtainable.
They should be of all metal construction, and not with a wood casing, on account of
the danger from fire. They cost from twenty-five to fifty dollars.

An incubator may be extemporized by putting the bulb of an incandescent electric
lamp in a vessel of water. The proper temperature may be obtained by increasing
the amount of water or by covering the opening more or less completely with a towel.
The test-tubes to be incubated can be put into a fruit jar or tin can, which receptacle
is placed in the vessel heated by the lamp.

Emery suggests the use of a Thermos bottle as an incubator.

The vacuum bottle should be first warmed by pouring in warm water. After-
ward the bottle should be three-fourths filled with water at ioo°F.

Schrup suspends his cultures and thermometer in the water by threads attached
to pins in the cork of the vacuum bottle. The plug should be paraffined or covered
with a rubber cap. As regards the matter of a low-temperature incubator (for
gelatin work), this may be met by using a small refrigerator. The ice in the upper
part maintains an even cold, and by connecting up an electric lamp in the lower part
of the refrigerator we can easily maintain a temperature which only varies one or two
degrees during the twenty-four hours. The gelatin plates or tubes should be placed
on the shelves usually provided with the refrigerator and not on the bottom.

With a i6-candle-power lamp a temperature of about 2S°C. is maintained (this is
too high, being about the melting-point of gelatin); with an 8-candle-power, one
about 21° to 23°C.; and with a 4-candle-power, from 18° to 2o°C.; the box being
about 20X30X36 inches.

More recently we have used with entire satisfaction a low tempera-
ture incubator made by Hearson.

The low temperature is supplied by water from cracked ice packed in a large cen-
tral chamber. A small dynamo controlled by a thermostat circulates the water

i6

APPARATUS

around the chamber containing the gelatin cultures. It requires some time for
proper adjustment, but afterward maintains a uniform temperature. Should the
temperature of the room in which the incubator is installed be below 22°C. there is
provided an automatically controlled heating coil which operates when the surround-
ing temperature is lower than 22°C.

Centrifuge. — When much serum reaction work is done, an electrically run centri-
fuge is an absolute necessity. It should be strongly constructed and placed on a
firm base. There should be places for 8 tubes and the outer shell or guard should

FIG. 7. — i, 2, 3, Drawing put glass tubing; 4, 5, Wright's rubber bulb capillary
pipettes showing grease pencil mark for making dilutions; 6, 7, Wright's U-tubes;
8, p, 10, methods of drawing out test-tubes for vaccines in opsonic workj'n, bac-
teriological pipette.

be so strong that in event of the breaking of a tube, while the centrifuge is re-
volving at high speed, there would be no danger for the operator. Water-power-
driven centrifuges are less satisfactory and hand ones least so.

An electric drying oven is very useful, taking the place of a water
bath. In reactions like that for blood acidosis, where the steam vapor
interferes, it is almost essential.

Filter Pump.— A filter pump attached to the water faucet, preferably by screw
threads, is almost indispensable for filtering cultures, etc., and for cleaning small
pipettes, especially the haemocytometer pipettes. Such a filter or vacuum pump with

PIPETTES 17

a vacuum gauge is more easily controlled. In washing red cells in Wassermann
reactions a pipette attached to the rubber tubing of the pump facilitates the removal
of the supernatant fluid.

The filter pump is indispensable when using the various types of porcelain or
Berkefeld filters. The Punkal or Muencke types of filter are the most convenient
in filtering toxins or in the sterilization of certain media when heating would be
unadvisable.

-L-AVERY-

FIG. 8. — i, Apparatus combining various methods for culture of anaerobes; (a)
Hofmann clamp for connecting with vacuum pump; (6) pyrogallic at bottom of
bottle for Buchner's O absorption method; (c) deep glucose agar stab covered with
sterile liquid petrolatum (see anaerobes). 2, One-fourth inch capillary loop U tube
for making two nitric acid albumin tests (see chemical examination of urine). 3,
Piece of tubing bent to hold slide for steaming smears in flame. 4, Schmidt's fer-
mentation apparatus, as modified by using graduated cylinder (see under faeces).
5, One-fourth inch glass tubing, 4^ inches long with ^corks at each end. For
centrifuging faeces for ova. 6a, Apparatus connected with sterile centrifuge tube
for taking blood from vein of man or a guinea-pig or rabbit's heart. 6b, Erlenmeyer
flask which can be used instead of centrifuge tube. See under sections Immunity
and Blood. 7, A graduated pipette with Hofmann clamp applied to rubber bulb
for precise delivery of measured quantities of liquids.

Capillary Pipettes. — With the possible exception of the platinum
loop, there is no piece of apparatus so applicable to many uses as the
capillary pipette made from a piece of glass tubing.

These may be made in a great variety of shapes. The one with a hooked end, the
Wright tube, is the best apparatus for securing blood for serum tests. The crook
hangs on the centrifuge guard and by filing and breaking the thicker part of the tube
2

1 8 APPARATUS

the serum is accessible to a capillary rubber bulb pipette or to the tip of a haemocy-
tometer pipette. In this way dilutions of serum are easily made.

Quite recently I have been using the blood tube recommended by LYON. To make
it heat a 5- or 6-inch section of H inch tubing in the center and draw out as for
making 2 bacteriological pipettes. Divide and seal off the large end in the flame.
Next seal off the capillary end. Then apply a very small flame to a point on the
large end just before it begins to taper to the capillary part. The heat causes
the heated sealed off air inside to force out a blow hole. To use: Break off the
sealed capillary end and allow the capillary end to suck up blood from a drop just
as with the Wright tube. I consider this tube superior to the Wright one.

• Pipettes.— The capillary pipette is made by taking a piece of J

soft German glass tubing, about 6 inches long, and heating in the middle

in a Bunsen flame, revolving the tubing while heating it.

When it becomes soft in the center, remove from the flame and with a steady even
pull separate the two ends. The capillary portion should be from 1 8 to 20 inches in
length. When cool, file and break off this capillary portion in the middle. We then
have two capillary pipettes. By using a rubber bulb, such as comes on medicine
droppers, we have a means of sucking up and forcing out fluids by pressure with the
thumb and forefinger of the right hand. The bulb should be pushed on about H
to Y± inch; this gives a firmer surface to control the pressure on the bulb.

A bacteriological pipette is made by drawing out a g-inch piece of tubing about
3 inches at either end, then heating in the middle we draw out and have two
pipettes similar to the one shown in the drawing. A piece of cotton is loosely pushed
in just above the narrow portion. These may be wrapped in paper and sterilized for
future use. They may be made perfectly sterile at the time of drawing out.

Illustrations are given for apparatus for culturing spirochaetes as well as for
anaerobic plating methods in Fig. 6. In the same cut is shown an apparatus for
filling vaccine ampoules and also our aerobic plating method for class work.

Where gas is not at hand, the Barthel alcohol lamp gives a flame similar to that
of the Bunsen lamp and is equally satisfactory for heating glass tubing. By making
a collar with a lateral opening to fit the burner of a Primus lamp a powerful side-
flame is obtained which is almost as suitable for glass blowing as the Bunsen blast
usually employed.

The ordinary blast lamp used by plumbers is useful.

CHAPTER II
CULTURE MEDIA

WHILE there are certain advantages in sterilizing the glass test-tubes
prior to filling them with media, yet this may be dispensed with — the
sterilization after the media has been tubed being sufficient. If a dress-
ing sterilizer is at hand, this is preferable for sterilizing such media as
bouillon, potato, and agar (10 to 15 pounds' pressure for fifteen min-
utes) . Milk should be sterilized with the Arnold, subjecting the media
to three steamings for twenty minutes on three successive days. Gela-
tin may be sterilized in either way, but preferably in the autoclave at
7 pounds' pressure for fifteen minutes. As soon as taken out of the
sterilizer it should be cooled as quickly as possible in cold water. This
procedure tends to prevent the lowering of the melting-point of the
finished gelatin and also preserves its spissitude.

Blood-serum is preferably solidified as slants in a blood-serum inspissator. This
requires one to two hours. The subsequent sterilization in the autoclave or Arnold
should not be done immediately after making the solidified slants, but on the subse-
quent day. If done on the same day, many of the slants are ruined by being dis-
rupted by bubbles. The preparation of blood-serum slants or slants of egg media
can be conveniently carried out in a rice cooker (double boiler). Place the tubes in
the inner compartment of the cooker, obtaining the slant desired by manipulating
an empty test-tube, or with a towel or cotton batting on the bottom. Then cover
the tubes with another towel. The outer compartment should contain water alone
(not 25% salt solution). The inner compartment should be weighted down so that
it is surrounded by water — the light tubes not being sufficient to sink it. Allowing
the water in the outer compartment to boil one or two hours will inspissate or solidify
the slants satisfactorily. The sterilization on subsequent days may be carried out
in the same apparatus, although it is more efficient if done in an Arnold or an auto-
clave. (This sterilization in the rice cooker makes the media too dry.)

Rice Cooker. — In making media a rice cooker is almost essential; at any rate, it is
so if ease, expedition, and unfailing success in preparation are to be achieved. As it
is necessary to make the contents of the inner compartment boil, the temperature of
the water in the outer compartment must be raised. This is done by using a 25%
solution of common salt or a 20% solution of calcium chloride in the outer compart-
ment instead of plain water. Should CaCl2 be carried over to media in inner com-
partment (as by thermometer) coagulation of albumin and clearing of medium will
be prevented.

19

20 CULTURE MEDIA

Makers of apparatus for the bacteriological laboratory now furnish a vessel for
making media which has two bottoms with an intervening air space. This hot
air layer prevents the scorching of the media as is so liable to occur when a plain
saucepan is used. Advantages over the rice cooker are that time is saved in bringing
the media to a boil, and also in the maintaining of a brisk boiling temperature.

A 15% solution of salt raises the boiling-point 2^2°C.; a 20%, 3H°C., and a
25%, 4K0C. The raising of the boiling-point by calcium chloride is about the
same for similar strength solutions.

Although the Bacteriological Committee of the A. P. H. Association recommends
special steps to be taken in the preparation of gelatin and agar, yet for clinical pur-
poses it will be found satisfactory to keep on hand a stock of bouillon, and when it is
desired to make agar or gelatin to simply prepare such media from the stock bouillon
in the way to be subsequently given.

NUTRIENT BOUILLON

This may be made either from fresh meat or from meat extract.
Media from fresh meat are usually lighter in color and possibly clearer.
In the Philippines, however, certain measures employed for the preser-
vation of the meat made it very difficult to prepare clear bouillon
from it, so that meat extract was used entirely. There is very little
difference, if any, in the nutritive power of media made in either way.

The chief objections to fresh meat as a base are: i. It takes more time and trouble.
2. The reaction, due to sarcolactic acid and acid salts, is quite acid, so that it is
necessary to titrate and neutralize the excess of acidity. 3. The reaction of the
finished media tends to change unless the boiling at the time of making was very
prolonged. 4. It is not infrequent to have a heavy precipitate of phosphates
thrown down at the time of sterilization, thus making it necessary to repeat the
process of nitration and sterilization.

If fresh meat is used, take about 500 grams (i pound), remove fat and cut it
up with a sausage mill or purchase the meat already cut up as for a Hamburg steak.
It makes little difference whether the amount be 100 grams more or less. Place
the chopped-up meat in a receptacle and pour 1000 c.c. of water over it. Keep in
the ice chest over night and the next morning skim off with a piece of absorbent cot-
ton the scum of fat; then squeeze out the infusion with a strong muslin cloth, mak-
ing the amount up to 1000 c.c. This meat infusion contains all the albuminous mate-
rial necessary for the clarification of the bouillon. It is convenient to designate this
meat base as Meat Infusion to distinguish from the base containing meat extract.

Having obtained 1000 c.c. of this 50% meat infusion, we dissolve in it i% of
Witte's peptone and K% of sodium chloride. While there is a sufficiency of the vari-
ous salts necessary for bacterial development in the meat juices, yet there is not
enough to give the best results when bouillon cultures of various organisms are used
for agglutination tests; and furthermore, when bouillon is used for blood cultures,
disintegration of the red cells, with clouding of the clear medium, may occur if there
t>e not sufficient salt present to prevent this.

REACTION STANDARDIZATION 21

The salt and the peptone are best put in a mortar, and adding about i ounce
of the meat infusion we make a pasty mass; then we gradually add the remaining
infusion until solution is complete. It is sometimes recommended to use a tempera-
ture of so°C. to facilitate the solution of the peptone. This is not necessary, and
if the temperature is not watched closely it might go up to 65°C. or higher and we
should lose the clearing albuminous material from its coagulation. Of this rather
cloudy solution take up 10 c.c. with a pipette and let it run out into a porcelain dish.
Add 40 c.c. of distilled or rain water and about six drops of a 0.5% phenolphthalein
solution. (Phenolphthalein, 0.5; dilute alcohol, 100 c.c.) Bring the contents of
the porcelain dish to a boil and continue boiling for one or two minutes in order to
expel all CO2. Now from a burette filled with decinormal sodium hydrate solution,
run in this solution until we have the development of a faint but distinct pink in the
boiling diluted bouillon which is not dissipated on further boiling.

It is more satisfactory to take burner from beneath the porcelain dish just before
running in the N/io solution, again boiling so soon as a pink color is obtained. Hav-
ing obtained the light pink coloration we read off the number of c.c. or fractions of a
c.c. of N/io sodium hydrate solution added to produce the color. This number
gives the acidity of the bouillon in percentage of N/i acid solution.

Percent acid means that so many c.c. of N/i acid added to 100 c.c.
of the medium at the neutral point would give that percentage reaction.
Thus ij-^ c.c. of N/i HC1 solution added to 100 c.c. of medium at o,
would give us ij^% of acidity or + 1.5. (Accurately 98^2 c-c-)

Percent alkaline means so many c.c. of N/i sodium hydrate solution added to
100 c.c. of the medium at the neutral point. Thus a %% alkaline medium would be
one whose alkalinity would correspond to the addition of ^ c.c. of N/i NaOH to 100
c.c. of the medium at o. It is written —0.5.

If we took 100 c.c. of the medium and put it in a beaker and then ran in N/i
NaOH solution from a burette, it will be readily understood that if we had to add
3^2 c.c. of N/i NaOH to obtain the pink color, it would show that the acidity of the
100 c.c. of medium, being tested, corresponded to 3.5 c.c. of N/i acid solution,
and that its acidity was equal to 3^% of N/i acid solution, or that its reaction was
+3-5-

As N/i NaOH solution is too corrosive for general use in a burette, and as 10 c.c.
of medium is more convenient to work with than 100 c.c., we use a solution one-
tenth the strength of the N/i NaOH and we take only one- tenth of the 100 c.c. of
medium. In this way it is the same from a standpoint of directly reading off our
percentage reaction as if we had 100 c.c. of medium and used N/i NaOH solution.
The A. P. H. Association recommends 5 c.c. of the medium and the use of N/20
NaOH. As the N/io NaOH is always at hand for titrating gastric juice, the N/io
is used instead.

Should it be found difficult to carry on the titration while boiling the end reaction
may be fairly accurately determined in the cold. Deliver into a beaker from a
pipette 10 c.c. of the bouillon and make up to 50 c.c. with distilled water and add 5
drops of 0.5% phenolphthalein solution. Then run in N/io NaOH from a burette
and continue to add the N/io NaOH solution from the burette, drop by drop, until

22 CULTURE MEDIA

the addition of a drop fails to show any intensifying of the purplish-violet color at the
spot where it came in contact with the diluted bouillon in the beaker. This marks
the end reaction. A reaction of about +0.7 in the cold gives a delicate pink with
phenolphthalein as an indicator. Titration in the cold is not very satisfactory with
gelatin and agar.

Having determined the percentage acidity of the 10 c.c. sample tested, we easily
calculate the number of c.c of N/i NaOH solution required to be added to the 1000
c.c. of bouillon to obtain a reaction corresponding to the neutral point of phenol-
phthalein. It is more exact to take the average of two titrations.

As TOO c.c. of medium would require 3^ c.c., 1000 c.c. would require 10 times as
much, or 35 c.c. N/i NaOH solution. Having measured out and added 35 c.c.
of the N/i NaOH solution to the meat infusion, containing salt and peptone, we
have a solution which is exactly neutral to phenolphthalein, or o. It is usually con-
sidered that a reaction of about i% acid is the optimum reaction for bacterial
growth. Hence we should now add i% of N/i HC1 solution to the medium. This
would be accomplished by adding 10 c.c. of N/i HC1 solution to the 1000 c.c. of
neutralized medium, and we would have a medium with a reaction of -f-i. If we
desired a reaction of i% alkalinity we would add an additional c.c. of N/i NaOH
solution to every 100 c.c. of the medium at o, or 10 c.c. for the 1000 c.c. of medium.
The reaction would then be — i.

As a matter of convenience, we usually determine the reaction of the
medium, which is always more or less acid, and then add enough N/i
NaOH to reduce the acidity to the percentage we desire to set the med-
ium, instead of neutralizing all the acidity present and then, in a second
operation, restoring the acidity to the point desired.

Thus finding the acidity of the medium to be 3^% and desiring to give it an
acidity of i%, we would add only 2% c.c. of N/i NaOH to every 100 c.c. of medium,
or 25 c.c. for the 1000 c.c. of medium. The reaction would then be found to
be + i.

The neutral point of litmus is not a sharp one, but it corresponds rather closely
with a reaction of +1.5 to phenolphthalein. The recommendations of the A. P. H.
Association call for making the titration with the medium boiling. If the color of the
end reaction at boiling-point be obtained, it will be found that when cool it deepens
until it corresponds to the rich violet-pink of the end reaction in the cold or vice versa.

To summarize:

Take Peptone 10 grams

Sodium chloride. 5 grams

50% meat infusion 1000 c.c.

Dissolve the peptone and sodium chloride in the meat infusion and
add enough N/i NaOH to make the reaction +i.

Put the solution in the inner compartment of a rice cooker and bring to the boiling-
point and maintain this temperature for twenty minutes. The calcium chloride or

SUGAR BOUILLONS 23

sodium chloride in the outer compartment of the rice cooker enables us to secure
a boiling temperature for the contents of the inner compartment. Do not stir the
bouillon that is being heated, as the pultaceous membranous mass of coagulated
albumin makes filtration easy. Filter. The filter-paper in the funnel should be
thoroughly wet with water before pouring on the bouillon. This is to prevent
clogging of the pores of the filter-paper. Make up the quantity of filtrate to 1000
c.c. by adding water.

If greater exactness is demanded than answers for ordinary, clinical work, it is
advisable to again titrate and again adjust the reaction or to simply record the exact
reaction. It is more convenient to have a counterpoise to balance the inner com-
partment and then to add water to the medium until a kilo weight, in addition to the
weight balancing the container, is just balanced. Then titrate, adjust the reaction
(if so desired), and filter. Sterilize in the autoclave at i i5°C. for fifteen minutes or in
the Arnold on three successive days. The use of a balance is preferable in the
preparation of bouillon, necessary in making gelatin and imperative in making
agar media.

BOUILLON MADE FROM LIEBIG'S MEAT EXTRACT

Place in a mortar 3 grams of Liebig's extract, 10 grams of peptone and 5 grams
of sodium chloride. Dissolve the whites of one or two eggs in 1000 c.c. of water.
Then add this egg-white water, little by little, to the extract, peptone, and salt in the
mortar until a brownish solution is obtained. Pour this into the inner compartment
of a rice cooker; apply heat to the outer compartment containing the salt or calcium
chloride solution, allow to come to a boil and to continue boiling for fifteen to twenty
minutes. Do not stir. Place inner compartment on the scales and its counterpoise
and a one-kilo weight on the other side. Add water until the two arms balance.
Filter and sterilize.

The reactions of media made with Leibig's meat extract rarely exceeds +0.75
(from -f 0.6 to +0.9). Consequently for growing bacteria it is unnecessary to
titrate and adjust reactions unless precision is demanded.

SUGAR-FREE BOUILLON

Inoculate nutrient bouillon in a flask with the colon bacillus. Allow to incubate
at'37°C. over night. Pour the contents into a sauce-pan and bring to a boil to kill
the colon bacilli. Put about 15 grams of purified talc (Talcum purificatum, U. S. P.)
in a mortar. Add the dead colon culture, stirring constantly. Then filter through
filter-paper. It may be necessary to again pass the filtrate through the same filter
until the sugar-free bouillon is perfectly clear.

For ordinary purposes the very small amount of sugar in bouillon made from
Liebig's meat extract may be neglected in determining gas production; so that under
such conditions the various sugars could be added directly to the meat-extract bouil-
lon. Dunham's peptone solution may be used as a substitute for sugar-free bouillon,
the sugars being added to it. We prefer the serum water medium of Hiss.

24 CULTURE MEDIA

SUGAR BOUILLON

The sugar media ordinarily used for determining fermentation or gas production
are those of glucose and lactose. In special work such carbohydrates as saccharose
and maltose are used. The alcohol mannite is used in differentiating strains of
dysentery bacilli.

To make, simply dissolve i or 2 % of the sugar in sugar-free bouillon or that made
from meat extract. Tube in Durham's or the ordinary fermentation tubes and
sterilize in the autoclave at only about 5 pounds' pressure for fifteen minutes, or in
the Arnold. Ordinary peptone solution is a good substitute for sugar-free bouillon.

Too high a degree of heat may turn the sugar bouillon brownish. The nature
of the sugar itself may further be affected by too high a temperature. Many of
the carbohydrates added to bouillon are liable to be split up on subjection to any
marked sterilization. Maltose is particularly unstable. It is best to make about
20% solution of the carbohydrates in distilled water and sterilize in small flasks adding
enough to the sterile bouillon to give a i % solution. Inulin usually contains resist-
ant spores so that its sterilization may need the autoclave. One of the great diffi-
culties about reporting on sugar reactions is the possibility of not working with a
chemically pure sugar as well as with one changed by too much heat. Inulin is a
polysacchoride resembling starch but does not give the iodine reaction. It is
obtained from the roots of chicory or dandelion. For further notes on carbohydrates
see water analysis.

BESREDKA'S EGG BOUILLON

ioo c.c. broth without salt; 80 c.c. 10% egg-white solution; 20 c.c. 10% yolk of
egg solution.

The egg-white solution is prepared by constant heating while adding distilled
water little by little. It is then filtered through absorbent cotton and heated to
ioo°C. Then filter through filter-paper.

This opalescent liquid is then put into tubes or flasks and sterilized at ns°C. for
twenty minutes.

To ioo c.c. of -10% emulsion of egg yolk in water you add about i c.c. of N/i
NaOH solution. One should add enough of this caustic soda solution to clarify the
emulsion slightly; it should still be opaque when in a rather thick layer. Heat to
ioo°C., then filter and finally sterilize at ii5°C. for twenty minutes.

The bouillon is a 50 to 75% meat infusion with peptone.

This is an excellent medium for growing pneumococci, streptococci, meningococci
and gonococci; even the organism of pertussis will grow on this medium. Of course
such organisms as the typhoid-colon group, cholera, diphtheria and pathogenic
anaerobes grow readily on it.

The medium is particularly recommended for growing tubercle bacilli. For this
purpose the peptone in the bouillon is omitted as it is found that tubercle bacilli
grow better without it. Besredka found that tubercle bacilli grew better when he
omitted the addition of glycerine to his medium.

It will be noted that for tubercle bacilli the medium should contain neither pep-
tone, salt nor glycerine.

In the course of two to three weeks we have a thick membranous growth.

INDOL PRODUCTION 25

It is stated that after a growth of from four to six weeks human strains show a dry
scale-like growth which is easily detached from the sides of the flask, while bovine
cultures show a sort of mucoid growth adhering to the walls.

Egg Broth. — This has been extensively used in the war zone for growing anaerobes.
Emulsify one whole egg in 300 c.c. water. Bring slowly to a boil with frequent
shaking. Then tube and sterilize. The organism of malignant cedema grows par-
ticularly well in it.

CALCIUM CARBONATE BOUILLON

Where we wish to cultivate such organisms as streptococci and pneumococci in
massive cultures we may add small fragments of marble (calcium carbonate) so
that any inimical excess of acid may be neutralized. North used a glucose bouillon
containing calcium carbonate in the production of massive cultures of B. bulgaricus.

GLYCERINE BOUILLON

Add 6% of glycerine to ordinary bouillon. It is chiefly used in the cultivation of
tubercle bacilli.

PEPTONE SOLUTION (DUNHAM'S)

Dissolve i% of Witte's peptone and K% of sodium chloride in distilled water.
Filter, tube, and sterilize. Peptone soluTioiTmay be used as a base for sugar media
instead of bouillon. If is the«medium used in testing for indol production. This
test is made by adding from 6 to 8 drops of concentrated H2SO4 to a twenty-four- to
forty-eight-hour-old peptone culture of the organism to be tested. If the organism
produces both indol and a nitroso body, we obtain a violet-pink coloration, "cholera
red." If no pink color is produced on the addition of the sulphuric acid, add about
i c.c. of an exceedingly dilute solution (i : 10,000) of sodium nitrite.

Cholera Red. — It is very important in determining the "cholera red" reaction
to know that the peptone used will give the reaction as it is not given by true
cholera strains with certain samples of peptone.

For the Voges-Proskauer Reaction. — Fill fermentation tubes with a 2% glucose
Dunham's peptone solution and sterilize. After inoculation with the organism to be
tested incubate for three days. Then add 2 to 3 c.c. of strong caustic potash
solution. The development of a pink color on exposure to the air is a positive reac-
tion (the color of a weak eosin solution).

Hiss' SERUM WATER MEDIUM

Take one part of clear beef €erum and add to it about three times its bulk of
water. Heat, the mixture in the ?Ernold for fifteen minutes to destroy any diastatic
ferment which might be present. Color to a deep transparent blue with litmus
solution and then add i % of any of the various sugars used in fermentation tests.
Sterilize in the Arnold by the fractional method.

26 CULTURE MEDIA

NUTRIENT AGAR

In making agar medium it is preferable to use powdered agar, as this goes into
solution more readily than the shredded agar. The reaction of agar is slightly alka-
line, so that if 1 3^ to 2% of agar is added to nutrient bouillon having a reaction
of +i the finished product will be found to be about +0.8.

To make: Weigh out 15 to 20 grams of powdered agar and place in a mortar.
Make a paste by adding nutrient bouillon, little by little, and when a smooth even
mixture is made, pour it into the inner compartment of a rice cooker and add the
remainder of the 1000 c.c. of bouillon. The use of the balance is preferable.

The outer compartment of the rice cooker should contain the 25% salt solution.
Bring to boll, and the agar will be found to have entirely gone into solution after
five to ten minutes of boiling.

Then, using a funnel which has been heated in boiling water and which contains
a small pledget of absorbent cotton, or the cotton between two layers of gauze, we
filter the agar, tube it, and sterilize it in the autoclave or Arnold. One and one-half
percent agar can be readily filtered through filter-paper and gives a clearer medium.

Some prefer to place the filter stand, funnel with gauze cotton filter and flask in
an Arnold sterilizer, for twenty minutes, so that when taken out the funnel will not
only be hot but the filter, being moist, will allow of more rapid filtration.

By taking of meat extract 3 grams, peptone 10 grams, salt 5 grams,
powdered agar 15 grams, the white of one egg and 1000 c.c. of water,
making at first a paste of all the ingredients in a mortar, then gradually
adding the remainder of the 1000 c.c. of water,*putting in the rice cooker,
bringing to a boil without stirring, allowing to boil fifteen minutes and
then filtering through absorbent cotton placed between two layers of
gauze in a hot funnel, we obtain a satisfactory medium, the reaction of
which will be from +0.7 to +0.9. It is very important not to interfere
with the pultaceous coagulum which forms on the surface of the boiling
agar.

Where very exact adjustment of the reaction of the finished product is desirable
the method of preparation of the Committee on Water Analysis of the American
Public Health Association is to be preferred.

Dissolve 15 grams of agar in 500 c.c. of water in the inner compartment of the
rice cooker previously described. After the agar is in solution (after ten to fifteen
minutes boiling) remove the inner compartment, containing the 3 % agar solution, and
allow it to cool to about SS°C. Mix in the mortar, as described in the directions for
making nutrient bouillon from Liebig's extract, 3 grams of Liebig's extract, 10 grams
of peptone and 5 grams of sodium chloride in 500 c.c. of water containing the whites of
one or two eggs. Heat this mixture to 50 to S5°C^kid pour it into the agar solution,
in the inner compartment, which has been cooled to about 55°C. Now titrate this
mixture containing 500 c.c. of double strength agar and 500 c.c. of double strength
peptone, meat extract and salt solution. The resulting 1000 c.c. gives i^% agar
and i% peptone solution. Having adjusted the reaction by the addition of the

GONOCOCCUS STARCH MEDIUM 27

necessary amount of N/i acid or alkali, we place the inner compartment in the outer
one of the rice cooker, bring to a boil and filter through filter-paper which has been
wetted with boiling water. The filtration can be carried out in the autoclave or in an
Arnold sterilizer. Of course the ordinary filtering through gauze and cotton will
answer where clearer media is not an object.

GLUCOSE AGAR

Add the agar to i or 2% glucose bouillon and pro eed as for ordinary agar. If
preferred, the glucose agar can be made by rubbing up meat extract 3 grams, peptone
10 grams, salt 5 grams, glucose 10 grams and 15 grams of agar in 1000 c.c. of water
containing the white of egg (one to two eggs), then boiling in the rice cooker and
filtering.

GLYCERINE AGAR

Add the agar to 6% glycerine bouillon instead of nutrient bouillon, or the gly-
cerine may be added to nutrient agar which has been melted. Glycerine agar with a
reaction of o makes an excellent base for blood and serum media for use in culturing
delicate pathogens.

GLYCERINE AGAR EGG MEDIUM

Take the white and the yolk of one egg and mix thoroughly in a vessel kept
between 45° and 55°C. with an equal amount of glycerine agar. Tube the medium,
inspissate in a rice cooker as for serum tubes, and sterilize as for blood-serum tubes.

This makes an excellent medium for growing tubercle bacilli. As egg medium has
a tendency to be dry, it is well to add i c.c. of glycerine bouillon to each slant before
autoclaving.

VEDDER'S STARCH MEDIUM

Macerate 500 grams of beef in 1500 c.c. of water in ice box over night. In the
morning filter into stew pan through doubled gauze. Bring fluid slowly to a boil and
add agar iH%- Boil for from twenty to thirty minutes.

Titrate and correct reaction to 0.2 to 0.7 acid. Check reaction to show faint blue
on red litmus.

Let cool to about 6o°C. and add two whole eggs, well beaten, to clarify. Bring
very quickly to a boil, then turn down flame and allow to simmer until a complete
coagulum forms.

Filter through gauze and cotton and add i % starch.

Let stand in Arnold for about forty-five minutes, shaking about three times during
this period, to distribute the starch.

Tube or flask, and sterilize in autoclave for fifteen minutes at 10 pounds. There
must be plenty of water of condensation and the agar must not exceed i>£%.

This is especially recommended for culturing gonococci. English workers have
used it in isolating meningococci from carriers. For this purpose the starch agar
has i% of glucose added to it and colored with litmus solution. The Meningococcus
acidifies the glucose while the M. Catarrhalis does not.

28 CULTURE MEDIA

NUTRIENT GELATIN

Place in a mortar 3 grams of Liebig's extract, 10 grams of peptone and 5 grams
of sodium chloride. Dissolve the whites of one or two eggs in 1000 c.c. of water.
Then add this egg-white water, little by little, to the meat extract, peptone and salt,
in the mortar, until a brownish solution is obtained. Pour this into the inner com-
partment of the rice cooker and bring the temperature up to 45°C. (This prelim-
inary elevation of temperature is better carried out in some heated water in a pan,
as the heating by means of the salt solution in the outer compartment of the rice
cooker is difficult to control, so that a temperature approximating 7o°C. might be
obtained and the albumin of the white of egg coagulated. The temperature in
the outer compartment might be approaching boiling before the contents of the inner
compartment would show 45°C.) Now take about 120 grams of "gold label" or
other good quality gelatin (12%) and crush it down in the meat extract egg- water
solution in the inner compartment of the rice cooker.

The gelatin quickly goes into solution at 45°C. Gelatin being quite acid it will
probably be found upon titration that the reaction is about +4%. ' N/i NaOH
solution is added to bring the reaction to about +1% or 3 c.c. of N/i NaOH for
each 100 c.c., provided the reaction were exactly +4%- The procedure is the same
as for bouillon. The color reaction is not quite as distinct with gelatin as with
bouillon.

Having neutralized and allowed to boil for fifteen minutes, we filter through filter-
paper in a hot funnel. As it is very important that gelatin should be perfectly
clear, it is better to filter through filter-paper than through cotton. The filter-paper
should be very thoroughly wetted with very hot water before filtering gelatin or agar.

Tube the medium and sterilize, either in the Arnold on three successive days or in
the autoclave at 8-10 pounds' pressure for ten minutes. The tubes should be
cooled as quickly as possible in cold water after taking out of the sterilizer.

AGAR GELATIN MEDIUM (NORTH)

Lean chopped beef or veal 500 grams.

Agar 10 grams.

Gelatin, Gold label 20 grams.

Peptone, Witte's, 20 grams.

Sodium chloride 5 grams.

Distilled water, q.s •. 1000 c.c.

Extract the chopped beef with 500 c.c. distilled water for eighteen hours, strain
through muslin and combine the ingredients in the usual way. Adjust the reaction
to the neutral point, using phenolphthalein as indicator.

North states that this medium is excellent for streptococci, pneumococci and
diphtheria bacilli because it is soft, moist, and can be used at 37°C.

It is claimed to be of special value for carrying stock cultures. It is useful as a
plating medium for milk.

LITMUS MILK

Milk for media should be as fresh as possible. It should then be put in a 1000-
c.c. Erlenmeyer flask, sterilized for fifteen minutes in the Arnold, and set over night

AZOLITMIN 2Q

in the refrigerator. The next morning the milk beneath the cream should be
siphoned off. The short arm of the siphon should not reach the bottom of the flask
so as to avoid the sediment. Add sufficient litmus solution to this milk to give a
decided lilac tinge; tube and sterilize in the Arnold on three successive days.

Litmus milk which apparently is as satisfactory as the above as regards nutritive
quality and cultural characteristics can be made from certain canned milks which
have not been condensed or sweetened and which do not contain chemical pre-
servatives. The "Natura" brand of milk is the one I have experimented with.

Litmus Solution. — A simple solution may be made by digesting the powdered
cubes repeatedly with hot water, mixing the extracts, and, after allowing them to
stand all night, decanting the solution from the inert sediment into a clean bottle.

In litmus solution so made, however, a red dye is also present while calcium and
other salts are dissolved out. For bacteriological purposes a pure solution of the
blue dye should be used. This is called "azolitmin." It is freely soluble in water
but insoluble in alcohol.

It can be conveniently prepared as follows: Weigh out 2 ounces of powdered
litmus; digest repeatedly with fresh quantities of hot water until all the coloring
matter is dissolved out; allow to settle, and decant off the fluid from the insoluble
powder. Add together the extracts, which should measure about a liter. Evapo-
rate down the solution to a moderate bulk, then add a slight excess of acetic acid,
so as to convert all carbonates present into acetates. Continue the evaporation,
the later stages over a water bath, until the solution becomes pasty. Add 200 c.c.
of alcohol, and mix thoroughly. The alcohol precipitates the blue coloring matter,
while a red coloring matter, together with the alkaline acetate present, remains in
solution. Transfer to a filter. Wash out the dish with alcohol and add this to
the filter. Wash the precipitate on the filter with alcohol. Dissolve the pure color-
ing matter remaining on the filter in warm distilled water and dilute to 500 c.c.
Azolitmin solution prepared in this way is more sensitive than ordinary litmus
solution.

Azolitmin in powder can be purchased from dealers in chemicals.

POTATO SLANTS

Take Irish potatoes and scrub thoroughly with a stiff brush. Then pare off
generously all the outer portion. From the white interior cut out cylinders with a
cork borer. These cylinders should be of Y^ to % of an inch in diameter. Divide
a cylinder by a diagonal cut. This gives a plug with a flat base, the other extremity
being a slant. These potato plugs should be left in running water over night or
washed with frequent changes of water. This prevents the blackening of the plug.
Into a i-inch test-tube drop a pledget of absorbent cotton well moistened with water.
Then drop in the potato plug, base downward. Sterilize in the autoclave at 15
pounds for fifteen to twenty minutes, to insure sterility.

For glycerine potato, soak the plugs in 6% glycerine solution for about one
hour. Then drop a pledget of absorbent cotton moistened with the same glycerine
solution into the test-tubes and follow it with the potato plug. Sterilize in the
autoclave.

30 CULTURE MEDIA

BLOOD-SERUM

The blood of cattle should be collected in large pans or pails at the abattoir.
This vessel of blood should then be kept in the cold-storage room and the next morn-
ing the more or less clear serum will have been squeezed out from the clot. Collect
this serum and keep in the ice chest for future use. If to be kept for a long time, it
is advisable to add about 2% of chloroform to the serum in tightly corked flasks.
This will not only keep the serum, but will eventually sterilize it.

To make Loffler's serum, take i part of glucose bouillon and 3 parts of blood-
serum. Mix, tube, and coagulate the albumin in the inspissator or rice cooker,
giving the tubes a proper slant before heating. Sterilize the following day in tl
autoclave as previously directed (7 pounds) or in the Arnold on three successive da}

A SUBSTITUTE FOR ORDINARY BLOOD-SERUM

Add from 10 to 15 c.c. of i% glucose bouillon to the white and yolk of one egg,
make a smooth mixture in a mortar and tube. Inspissate and sterilize as for ordi-
nary serum slants. The morphology of the diphtheria bacilli and the luxuriance of
growth is similar to that of cultures on Loffler's serum.

When this medium is to be used for culturing tubercle bacilli add about i c.c.
of glycerine bouillon to each tube before final sterilization in the autoclave. The
cotton plugs should be paraffined to prevent drying of the slants in the incubator.
This medium seems to answer as a substitute for Dorsett's egg medium. (While
glycerine bouillon favors growth of human tuberculosis, it is not so satisfactory fc
bovine tuberculosis as plain glucose bouillon.) This is better than the various whit(
of egg substitutes usually recommended. (Pouring a little alcohol in the mort<
and moistening the sides by tilting, then burning off the alcohol, in a measure
sterilizes the mortar. If the egg is cracked open with a sterile knife, a medium can
be prepared which will be sterile as the result of the two-hour inspissation in the rice
cooker.) By covering the tube with a rubber cap or preferably, by heating the
plugged end of the test-tube, quickly withdrawing the cotton plug and dipping the
part of the plug which enters the tube into hot melted paraffin, then quickly reintro-
ducing the plug, the contents of the tube will be prevented from drying out. This
procedure is essential for growing tubercle bacilli.

Egg media are excellent for culturing anaerobes. One can add about 5 drops of
i% neutral red aqueous solution to each egg, inspissating as above. The reddish
color appears in colonies producing acid and is of value in anaerobic work to make
such colonies more distinct.

DORSETT'S EGG MEDIUM

This is prepared by breaking whole eggs into a sterile flask, mixing thoroughly then
adding 25 c.c. water to every 4 eggs, straining through a sterile cloth and tubing 10
c.c. quantities. These tubes are slanted in an inspissator and kept at 73°C. for
four or five hours on two successive days. On the third day a temperature of 76°C. is
applied. Before inoculating add 3 or 4 drops of sterile water to each tube. The
tuberculous material should be rubbed into the surface well and the plugs paraffined.

BLOOD AGAR 31

HYDROCELE, SERUM, ASCITIC AND MILK AGAR

To tubes of melted agar at 5o°C., add from i to 3 c.c. of hydrocele or ascitic fluid,
observing aseptic precautions. Allow the agar to solidify as a slant, or as a poured
plate.

For milk agar we add 2 or 3 c.c. of plain or litmus milk to a tube of melted agar.
This makes an excellent plating medium for B. bulgaricus. When poured into a
plate it is opaque but the colonies stand out well.

For obtaining sterile serum we generally use the apparatus described under
Blood Agar and as a rule take the blood from vein of arm in man or vein of neck of
sheep used for Wassermann work. The sterile serum separates from the clot and
can be pipetted off with a sterile pipette and added to the melted 2 to 3% agar as
above. A method recommended by Fildes is to take blood at the slaughter house
in sterile vessels, allow to clot and remove serum. Five c.c. of ether is added to
each 100 c.c. in a glass-stoppered bottle of which the stopper is fixed in and the mix-
ture heated for one hour in a water-bath at 45°C. after which it is placed in the
incubator at 37°C. for several days, by which time the serum is sterile. Before using
the ether is driven off at 45°C. This is better than the old method of sterilizing
with 2% chloroform.

BLOOD AGAR

For obtaining blood to make blood agar we use the apparatus described under
blood culturing and shown in Fig. 8. We take human blood from the arm vein,
sheep blood from the neck vein, or rabbit blood from the heart. In the Erlenmeyer
flask of 100 c.c. capacity is placed 5 c.c. of sterile 10% sodium citrate solution pro-
vided we want to take 50 c.c. of blood from sheep or man. The final mixture to
prevent coagulation should contain about i% sodium citrate. As a rule we only
take about 25 c.c. from man or rabbit so 2^ c.c. of 10% citrate would. suffice. The
perforated rubber stopper is removed and replaced with the sterile cotton plug of
the flask and the fluid mixture pipetted off and added to the melted agar.

Mixture is facilitated by rotating the tube rapidly between the hands. The
medium may be slanted or poured into plates. As this medium is satisfactory
for the growth of haemoglobinophilic organisms-,' as well as for others, we use it as a
routine plating medium, the others being nutrient agar and Endo.

BLOOD-STREAKED AGAR

Sterilize the lobe of the ear and puncture with a sterile needle. Collect the ex-
uding blood on a large platinum loop and smear it over the surface of an agar slant.
It is advisable to incubate over night as a test for sterility. Plates or slants of glycer-
ine agar of neutral reaction smeared with blood give the best results when such
delicate pathogens as pneumococci, streptococci, gonococci or meningococci are to
be cultured.

BILE MEDIA

Secure ox bile from the abattoir or human bile from cases of gall-bladder drainage
in hospitals. Put about 10 c.c. in each tube and sterilize. Some prefer to add

32 CULTURE MEDIA

i% of peptone. Conradi's medium is ox bile containing 10% of glycerine and 2%
of peptone. This is the medium for blood cultures in typhoid, etc.

The bile lactose medium now used in water analysis is made by adding i% of
lactose to ox bile and tubing in fermentation tubes. As a substitute for fresh bile
one may use a 15 to 20% solution of a good quality of inspissated ox gall (Pel Bovis
Purincatum). A liver bouillon made by using 500 grams of finely divided beef liver
in 1000 c.c. of water with i% peptone, and prepared as for meat infusion broth, is a
good substitute for bile.

RECTOR'S BILE LACTOSE NEUTRAL RED MEDIUM

This is recommended in the isolation of the colon bacillus as superior to lactose
litmus agar. It consists of 10% of dried ox bile, i% of peptone, and i>£% agar.
After the medium is filtered and tubed we add i% of lactose and i% of a i-ioo
neutral red solution. Colon colonies have a distinct purplish red zone. Further-
more the bile inhibits the growth of many organisms which give pink colonies on
lactose litmus agar. MacConkeys' bile salt medium contains %% oi sodium
taurocholate and is colored with neutral red.

THALMAN'S MEDIUM FOR THE GONOCOCCUS

Five hundred grams of lean, finely minced beef are placed in 1000 c.c. of distilled
water and allowed to stand over night in an ice box. It is then filtered and the fil-
trate made up to 1000 c.c. with distilled water. To 100 c.c. of the beef juice add
iM grams of agar, and boil for 15 minutes. Then add 2 grams of glucose, and bring
the reaction to plus 0.6 by addition of N/i NaOH. Tube, sterilize, slant, and in-
cubate over night. No peptone or salt is required. We now use Vedder's starch
agar.

PETROFF'S TUBERCLE BACILLUS MEDIUM

This is a remarkably valuable medium for isolating tubercle bacilli from sputum or
pus directly. Fresh sputum shouW be used and for destruction of contaminating
organisms it should be shaken up with an equal amount of 3% NaOH solution and
left in the incubator for one or two hours. Neutralize with N/i HC1, using litmus
paper, and centrifugalize. Take up sediment and smear out on slants of the
following medium.

Treat 500 grams of chopped up meat with 500 c.c. of 15% glycerine solution. Keep
in ice chest twenty-four hours and filter through gauze. Sterilize the shells of eggs
by immersion in 70% alcohol for ten minutes or by dipping them in boiling water
for five seconds or so. Mix white and yolk of these eggs in a sterile mortar and add
an equal volume of the glycerine meat infusion which should have added to it before
mixing i c.c. of i% alcoholic solution of gentian violet to each 100 c.c. of the
glycerine meat infusion.

Should one be culturing bovine strains the glycerine should be omitted from the
meat infusion but the gentian violet (1-10,000) added. Put 3 to 4 c.c. of this me-
dium in test-tubes and inspissate as slants at 85°C. until the medium is solidified.

ENDO'S MEDIUM 33

Subject these slants to temperature of 75°C. on the second and third days for one
hour.

PLATING MEDIA FOR FAECES WORK

The media of Endo, Conradi-Drigalski and the lactose litmus agar medium are
probably the most satisfactory of the numerous ones that have been proposed for
plating out faeces. A convenient way of preparing any one or all of these, and which
apparently gives media equal to that prepared according to the original formulae,
is as follows:

Liebig's extract 5 grams.

Salt 5 grams.

Pepton 10 grams.

Agar 30 grams.

Water to make 1000 c.c.

Prepare as for ordinary nutrient agar, with the difference that the reaction should
be brought down to o. Some prefer a reaction of +0.2.

A stiff agar (3%) is employed to check the diffusion of acid beyond the colony.

FOR ENDO'S MEDIUM

Keep this agar base in 100 c.c. quantities in Erlenmeyer flasks instead of test-
tubes. (If more convenient smaller quantities may be put in the flask.) When
needed for plating, melt a flask of this agar, and while liquid add to the 100 c.c.
6 drops of a saturated alcoholic solution of basic fuchsin, and then about 20 drops
of a freshly prepared 10% solution of sodium sulphite. The sulphite solution de-
colorizes the intense red of the fuchsin to a light rose pink. This color fades to a
light flesh or pale salmon color when cold. Now add 5 c.c. of a freshly prepared
hot aqueous 20% solution of chemically pure lactose. If only occasionally using
such media, tube in "20 c.c. quantities and add i drop of the basic fuchsin and 4
drops of the sodium sulphite solution and i c.c. of the hot freshly prepared lactose
solution to a tube of the melted agar base just before pouring the plate. This me-
dium contains i % of lactose. Kendall prepares an Endo medium which only con-
tains iH% of agar and with a reaction just alkaline to litmus (about plus 1.2%).

Colon bacilli show on this medium as vermilion colonies, which in about forty-
eight hours have a metallic scum on them. Typhoid and dysentery colonies are
grayish. Streptococci a deep red.

FOR LACTOSE LITMUS AGAR

Color the 100 c.c. of agar base with litmus solution to a lilac color. Then add
5 c.c. of the hot freshly prepared 20% lactose solution in distilled water. This
may be tubed, putting 10 c.c. in each test-tube, or put in quantities of 50 or
100 c.c. in small Erlenmeyer flasks. It is then sterilized in the autoclave (10
pounds for fifteen minutes) or in the Arnold.
3

34 CULTURE MEDIA

FOR CONRADI-DRIGALSKI MEDIUM

To ioo c.c. of lactose litmus agar add i c.c. of a solution of crystal violet (crystal
violet o.i gram, distilled water ioo c.c.). The medium is then ready to put into
plates. Colon colonies are pink. Typhoid and dysentery colonies, a bluish-gray.

CONRADI'S BRILLIANT GREEN MEDIUM

Take of Liebig's extract 20 grams (2%), peptone 10 grams (i%), agar 30 grams
(3%) and water to 1000 c.c. This amount of meat extract should give about the
proper acidity, +3. If not, the reaction should be adjusted to that point. Filter
through cotton, tube 150 c.c. amounts into 250 c.c. Erlenmeyer flasks and sterilize.

Then add i c.c. of a i to 1000 aqueous solution of brilliant green (Hochst) and
i c.c. of a i% solution of picric acid to the flasks containing 150 c.c. of the melted
agar. Sterilization after adding the dyes precipitates them and is unnecessary.
Pour the finished medium into large Petri dishes and inoculate the surface with the
faeces.

Brilliant green does not interfere with agglutination as does malachite green.

This medium is considered by some authorities the one of choice in isolating
typhoid bacilli from fasces and urine.

The surface of the poured plates of Endo, Conradi-Drigalski, and the brilliant
green media should be dried in the incubator before smearing with the fasces. For
routine work I prefer Endo's medium followed by Russell's double sugar agar.

SELECTIVE MEDIA FOR CHOLERA

Dieudonne's medium rests on the ability of cholera to grow when alkali is present
in such amounts as to inhibit the growth of other faecal bacteria.

Take equal parts of defibrinated blood obtained at the slaughter house and
normal NaOH solution. Mix 30 parts of this alkaline blood mixture with 70 parts
of hot 3% nutrient agar. The poured plates should be left half open over night in
the incubator otherwise even cholera will not grow on the plates.

Krumwiede has as a formula for his medium equal parts of whole egg and water,
to which 50% water egg mixture is added an equal amount of 12^% crystal sodium
carbonate solution. This alkaline egg mixture is steamed for twenty minutes. To
prepare add 30 parts of this alkaline egg mixture to 70 parts of meat extract free
3% agar. (No meat extract; only peptone and salt.) The cholera colony has a
hazy look, like a little wad of absorbent cotton sticking to the surface with a metallic
luster halo.

Other selective media for cholera are those of Kabeshima in which a haemoglobin
extract is treated with alkalis and added to agar.

The medium of Esch has been highly recommended. It is easy to make. Heat
500 grams chopped up beef with 250 c.c. normal NaOH solution in a pot and when
disintegrated filter through cloth and sterilize. About i part of this alkaline extract
is added to 2^ to 2 parts of agar. The plates must be dry. The transparency of
this medium is an advantage.

N. N. N. MEDIUM 35

RUSSELL'S DOUBLE SUGAR AGAR

A fairly stiff agar (2 to 3%) with a reaction of about plus 0.7 is colored with litmus
solution to produce a distinct purple- violet color. It may be necessary to add more
alkali. To this litmus tinted agar is added i% of lactose and 0.1% of glucose and
the medium as thus prepared is tubed and slanted. Sterilization should be carried
on in the Arnold, on two successive days, as the autoclave temperatures tend to
break up the sugars.

On these slants typhoid shows a delicate growth on the violet slant with a deep
pink in the butt of the tube. The paratyphoids show gas bubbles in a pink butt with
a violet slant.

The colon bacillus turns both slant and butt a deep pink and the butt is filled
with gas bubbles. To inoculate this medium we take material from a suspicious
colony grown on Endo and smear the material on the slant; then with the same plati-
num needle we stab into the butt.

Culture Media for Protozoa

MEDIUM OF MUSGRAVE AND CLEGG

Dissolve in 1000 c.c. of water 0.3 to 0.5 gram Liebig's extract and 0.3 to 0.5
gram of common salt. If desired for plating add 2 to 3% of agar.
A very satisfactory substitute is ordinary nutrient bouillon diluted one to ten.

MEDIUM OF SMITH

Glucose i.o gram; Peptone i.o gram; Nad 0.2; Aqua destill. 1000.0; Na2CO3 0.3.
Agar q. s. is added for solid medium.

MEDIUM OF CASTELLANI

This is an aqueous medium containing i% of lactose and 10% of egg albumin.
This may replace water of condensation in an agar slant.

AUTOLYZED TISSUE MEDIA

Couret and Walker have possibly grown amoebae from liver abscess on autolyzed
tissue media. Sterile organs from healthy animals as well as human placenta are
placed in sterile flasks and kept in a thermostat at 4o°C. for about two weeks. The
liquid from the autolyzed tissues should be about neutral and is either applied to
the surface of agar slants or is mixed with melted agar. The medium is then
sterilized.

Now MACNEAL MEDIUM

Cover 125 grams of chopped up beef with 1000 c.c. of water and place over night
in the refrigerator. Strain and add 20 grams of peptone, 5 grams salt, 10 c.c. of

36 CULTURE MEDIA

normal sodium carbonate solution and 20 to 25 grams agar. Prepare as for nutrient
agar and sterilize. To i part of this one-quarter strength meat infusion nutrient
agar, when melted and cooled down to 6o°C., add twice its volume of defibrinated
rabbit's blood. This medium is the standard one for the culture of certain trypano-
somes and other protozoa. Under the designation N.N.N. medium (Nicolle
Novy MacNeal) Nicolle has modified the medium so that there is only salt and agar
in the base to which the blood is added instead of one containing meat extract and
peptone. It is the Hb which seems essential in the culture of various protozoa.
Rogers used citrated salt solution, which was slightly acidified with citric acid, in
his culturing of Leishmania from the splenic blood of cases of kala azar. Incubation
at 22°C.

ROW'S H^EMOGLOBINIZED SALINE MEDIUM

Take 10 c.c. blood from rabbit's heart or arm vein of man, defibrinate the blood
and then add 10 volumes of distilled water to lake the cells (liberation of Hb). One
volume of this laked blood solution is added to two volumes of sterile 1.2% salt
solution.

CULTURE MEDIA FOR TREPONEMATA

I. Noguchi formerly first inoculated material containing treponemata into the
testicle of rabbits, obtaining by this procedure a pure culture, after a few transfers
to the testicles of other rabbits. He now grows the organism directly from serum
from a chancre. Test-tubes 2 by 20 cm. are filled with 15 c.c. of a medium consisting
of 2 parts of 2% slightly alkaline agar to which when melted and cooled down to
So°C. is added i part of ascitic or hydrocele fluid. At the bottom of the medium
in the tube is placed a fragment of fresh sterile tissue, preferably a piece of rabbit's
kidney or testicle. After the medium solidifies a layer of sterile paraffin oil is run
in so that it covers the solid medium to a depth of 3 cm. The material is inoculated
at the bottom of the tube with a capillary pipette. Incubation at 37°C. is carried
on for two weeks. The tissue acts by removing any oxygen that may be present in
the depths of the medium. Anaerobiosis is a necessary condition. Many specimens
of ascitic fluid are unsuited. The tubes of Noguchi and Bronfenbrenner are shown
in Fig. 6. Bronfenbrenner uses a iH% a8ar instead of the 2% used by Noguchi.

M'Leod and Soga have simplified Noguchi's method as follows: Take a test-tube
and fit a perforated rubber stopper which can be pushed down the tube. A piece
of glass tubing is passed through the stopper to project slightly into the test-tube.
The other end of the glass tube is drawn out into a capillary tube and bent over at
an acute angle. The test-tube is filled to K or % of its depth with neutral bouillon.
This is freshly boiled and when cool a piece of sterile tissue is dropped in. A strip
of sterile gauze is drawn through a glass bead and soaked in the material it is desired
to culture and dropped into the bottom of the tube alongside the fragment of sterile
tissue. Ascitic fluid is then run in to a point which would be reached by the bottom
of the rubber stopper. As quickly as possible push in the stopper and when the
fluid appears in the capillary tube seal off the end in a small flame. Material for
study can be obtained afterward by breaking off the capillary tip and introducing
a capillary pipette.

CULTURING TREPONEMATA 37

II. Serum Agar of Muhlens and Hofmann. — Fill sterile test-tubes one-third full
with horse serum. This is sterilized on three successive days at S5°C. Then
add an equal amount of a 3% agar containing 0.5% glucose which has been melted
down and cooled to 5o°C. The mixed serum agar is then kept at S5°C. for two hours.
Such tubes are inoculated as for ascitic agar rabbit tissue media and incubated under
anaerobic conditions, preferably in a flask from which the air has been exhausted and
the remaining oxygen absorbed as shown in the anaerobic bottle described and
illustrated in Fig. 8.

CHAPTER III
STAINING METHODS

IN order to study a bacterial or blood specimen the first essential is
a properly prepared film; the matter of staining is of less importance.

The slide or cover-glass, after cleaning with soap and water or by special solutions,
should be polished with a piece of old linen. If a glass surface is free of grease a
loopful of water will smear out evenly and over the entire surface. The only quick
practical way to make the slide or cover-glass grease free is to burn the surface for a
moment in a Bunsen or alcohol flame. The cover-glass must not be warped. To
make a preparation, apply a small loopful of distilled water on the slide or cover-glass
and, touching a colony with a platinum needle, stir the transferred culture into the
loopful (not drop) of water. The mistake is almost invariably made of taking up
too much bacterial growth. Fluid cultures do not need dilution. Smearing the
mixture over a large part of the cover-glass or over an equal area of a slide, it is
allowed to dry. If very little water is used, the preparation dries readily. Other-
wise it can be dried in the fingers high over a flame. As soon as dry, the cover-glass
should be passed three times through the flame, film side up, to fix the preparation.
Slides may be fixed by passing them five times through the flame, but the method by
burning alcohol recommended for fixing blood-films gives more satisfactory bacterial
fixation. For routine work the stain recommended is a dilute carbol fuchsin. Drop
about 5 to 10 drops of water on the cover-glass, then add i drop of carbol fuchsin.
Allow the dilute stain to act from one to two minutes, then wash in water, dry be-
tween small squares of filter-paper (4X4 inches), and mount in balsam or the oil
used for the K 2-inch immersion objective. LofBer's methylene blue is equally good
as a stain.

By far the best mounting medium is liquid petrolatum. This not only has the
advantage of always being of proper consistence for mounts, as opposed to Canada
balsam, which must frequently be made thinner with xylol, but it is less sticky and
does not develop the acidity which causes balsam mounts of Romanowsky stains
to fade. Furthermore, it has superior optical qualities. It is also applicable for
mounting small insects and sporangia of moulds. For permanent preparations the
border of the cover-glass should be sealed with gold size or some other cement.
Some prefer to mount directly in water without preliminary drying. It is good
practice to make a rule to always keep the smeared side of the preparations up —
never allowing it to be reversed. By this simple rule, preparations can be carried
through the most complicated staining methods without the necessity of scratching
the cover-glass, etc., to see which is the film side. In grasping a cover-glass with a
Cornet or Stewart forceps, be sure that the tips are well by the margin of the glass,
otherwise the stain will drain off. In staining with slides, the grease pencil and the

38

GRAM'S STAINING METHOD 39

glass tubing, as recommended under Blood Smears, will be found useful. The dilute
carbol fuchsin and Loffler's methylene blue are probably the best routine stains. As
a rule better preparations are obtained with dilute stains than with more concen-
trated ones.

Loffler's Alkaline Methylene Blue.— Saturated alcoholic solution
of methylene blue, 30 c.c.; i to 10,000 caustic potash solution, 100
c.c. (Two drops of a 10% solution KOH in 100 c.c. of water makes a
i : 10,000 solution.)

Carbol Fuchsin (Ziehl-Neelsen).— Saturated alcoholic solution basic
fuchsin, 10 c.c.; 5% aqueous solution carbolic acid, 100 c.c.

Gram's Method. — The most important staining method in bacteri-
ological technic and the one so rarely giving satisfactory results to the
inexperienced is Gram's stain. In using this method, the following
points must be kept in mind:

1. Laboratory cultures (subcultures) which have been carried over for years
frequently lose their Gram characteristics.

2. Cultures which are several days old or dead or degenerated do not stain char-
acteristically.

3. The aniline gentian violet deteriorates when exposed to light in two or three
days — it should be kept in the dark. It should have a rich, creamy, violet appear-
ance.

4. The iodine solution deteriorates and becomes light in color. It should be of
a rich port-wine color.

5. The decolorizing with 95% alcohol should stop as soon as no more violet stain
streams out. This is best observed over a white background, washing at intervals.
Do not confuse stain on forceps for that on preparation.

6. The preparation should be thin and evenly spread. Some prefer carbol
gentian violet to aniline gentian violet. (Saturated alcoholic solution of gentian
violet, i part; 5% aqueous solution of carbolic acid, 10 parts.) This tends to over-
stain.

The formula for aniline gentian violet is i part of saturated alcoholic solution
gentian violet and 3 parts of aniline oil water (made by adding 2 c.c. aniline oil to
100 c.c. distilled water, shaking violently for three to five minutes and then filtering
several times to get rid of the objectionable oil droplets which, in a Gram-stained
preparation, show as confusing black dots).

The following stock solutions of Weigert are recommended:

No. i No. 2.

Gentian violet. ...... 2 grams. Gentian violet. ... 2 grams.

Aniline oil 9 c.c. Distilled water — 100 c.c.

Alcohol (95%) 33 c.c.

These stock solutions keep indefinitely. Mix i c.c. of No. i with 9 c.c. of No. 2.
Filter. This keeps about two weeks and is the solution to pour on the preparation.
It may be kept on from two to five minutes. Some hasten the staining by steaming

STAINING METHODS

as for tubercle bacilli. Next wash the preparation with water and flood the cover-
glass with Gram's iodine solution. Some bacteriologists simply pour off excess of
aniline gentian violet and immediately drop on the iodine solution. It is well to
repeat the application of the iodine solution a second time.. The iodine solution is
left on one minute or until the preparation has a coffee-grounds color.

GRAM'S IODINE SOLUTION

Iodine i gram.

Potassium iodide 2 grams.

Distilled water 300 c.c.

After washing off the excess of iodine solution at the tap, drop on 95% alcohol
and decolorize until no more violet color streams out. Now wash again and counter-
stain either with the dilute carbol fuchsin or with a saturated aqueous solution of
Bismark brown.

The Gram-positive bacteria are stained a deep violet.

In staining smears of pus for gonococci or other Gram-negative bacteria it is best
to first stain with the gentian-violet solution for two to five minutes. Then wash
and examine the preparation mounted in water. The organisms stand out promi-
nently. After noting the presence of the cocci treat the smear with the Gram
solution and proceed as in the usual Gram staining technic.

STAINED BY GRAM'S METHOD
S. pyogenes aureus.
S. pyogenes albus.
S. pyogenes.
M. tetragenus.
Pneumococcus.
Anthrax bacillus.
Tubercle bacillus.
Lepra bacillus.
Tetanus bacillus.
Diphtheria bacillus.
B. aerogenes capsulatus.
Oidium albicans.
Mycelium of actinomyces.
Saccharomyces.
Hofmann's bacillus.
B. xerosis.

NOT STAINED BY GRAM'S METHOD
Meningococcus.
M. catarrhalis.
M. melitensis.
B. typhosus.
B. coli communis.
B. dysenteriae (Shiga).
Sp. cholerae asiaticae.
B. pyocyaneus.
B. mallei.

B. pneumonias (Friedlander).
B. proteus.
B. of influenza.
B. of bubonic plague.
B. of chancroid.
B. of Koch- Weeks.
Gonococcus.

Practically all pathogenic cocci are Gram-positive, except the Gonococcus, the
Meningococcus ', the M . catarrhalis, and the M. melitensis.

Practically all pathogenic bacilli are Gram-negative, except the spore-bearing
ones (exception B. malig. cedemat.}, the acid-fast ones, diphtheria and diphtheroid
organisms.

The bacillus of glanders is Gram-negative.

ACID-FAST STAINING 41

Method for Staining Acid-fast Bacilli. — i. Carbol fuchsin, with gen-
tle steaming for three to five minutes or in the cold for fifteen minutes.

2. Wash in water.

3. Decolorize in 95% alcohol containing 3% of hydrochloric acid
(acid alcohol), until only a suggestion of pink remains — almost white.

4. Wash in water.

5. Counters tain in saturated aqueous solution of methylene blue or
with Loffler's methylene blue.

6. Wash, dry, and mount.

The steaming of the slides with carbol fuchsin is most conveniently carried out
by resting the slides on a piece of glass tubing bent into a V or U shape.

The Leprosy Bacillus is usually considered as being rather easily decolorized by
alcohol. It is therefore often recommended to use 20% aqueous solution of sul-
phuric acid or nitric acid for decolorization instead of the acid alcohol above recom-
mended for tubercle bacilli. I have often found the leprosy bacilli as resistant to
alcohol as tubercle bacilli. The smegma bacillus, however, easily decolorizes with
the acid alcohol and in a well-decolorized smear from urinary sediment one can
usually feel sure that any acid-fast bacilli are tubercle bacilli.

Fontes Method. — A method in which the organisms or granules which stain by
the Gram method, and to which so much importance is attributed by Much, may
be stained, as well as those retaining acid-fast properties, has been proposed by
Fontes. The method is to stain the preparation with carbol fuchsin, decolorize
with acid alcohol, then carry through the various steps of the Gram method,
counterstaining, however, with Bismark brown. Fontes in his method used i part
of absolute alcohol and 2 parts of acetic acid as the decolorizing agent. I have
obtained, however, just as satisfactory results with the acid alcohol. By this
method the acid-fast tubercle bacilli show as red rods dotted with violet granules.
Those which do not fully retain acid-fast properties show as zigzag violet lines.

Herman's Stain for Tubercle Bacilli. — It has been claimed that this stain gives
better satisfaction than the Ziehl-Neelsen. It consists of two solutions: (i) am-
monium carbonate in distilled water, i%; (2) crystal violet (methyl violet 6B) in
95% ethyl alcohol, 3%. The two solutions are kept in separate bottles and, for
staining, i part of (2) is mixed with 3 parts of (i). The sections are placed on a
cover-glass, the water evaporated, and about 7 drops of the staining mixture
are placed on the specimen and allowed to steam for one minute over a water-bath.
Place for a few seconds in 10% nitric acid and then in 95% alcohol to decolorize.
Mount without a counterstain or use eosin i% or a very dilute fuchsin. The organ-
isms are purple. This staining method may be applied to smears of concentrated
or unconcentrated sputum in the same manner as for sections of tissue.

Smith's formol fuchsin :

Saturated alcoholic solution basic fuchsin 10 c.c.

Methyl alcohol 10 c.c.

Formalin 10 c.c.

Distilled water to make 100 c.c.

STAINING METHODS

This gives a very sharp differentiation of bacteria and nuclear structures. It
has a purplish tinge. Fixation by heat gives the best staining. Allow the stain to
act for two to ten minutes. It should not be used until after standing twenty-four
hours, and after standing about two weeks it appears to lose its sharp staining power

Archibald's Stain. — This is an excellent bacterial stain and has been
highly recommended by Blue and McCoy in plague work.

SOLUTION No. i.

Thionin 0.5

Phenol crys 2.5

Formalin i.o

Water 100.0

SOLUTION No. 2.

Methylene blue 0.5

Phenol crys 2.5

Formalin i.o

Water 100.0

Dissolve for twenty-four hours. Mix equal parts and filter. Stain smears fixed
by heat or otherwise for ten seconds.

Nicolle's Carbol Thionin

Sat. sol. thionin in 50% alcohol

Carbolic acid solution (2%).

10 c.c.
100 c.c.

Pappenheim's Stain. — Take a very small portion of methylene green on the point
of a penknife and shake it into a test-tube. Then take up twice as much pyronin and
deposit it in the same test-tube. Fill the test-tube one-half full with water and the
solution should have a distinct reddish-violet color. A drop on a piece of filter-paper
shows a violet center and peripheral green ring. The solution should be fresh.
Stain from two to five minutes. Differentiate with a little resorcin on a penknife
point dissolved in one-quarter of a test-tube full of alcohol. Dehydrate, clear and
mount. Polymorphonuclear nuclei stain greenish; nuclei of mononuclears and
plasma cells from bluish-red to dull violet. Cytoplasm of lymphocytes and plasma
cells purplish-red. Bacteria red.

Romanowsky Stains. — See under section on Blood. For mounting
specimens showing chromatin staining, as malarial parasites, trypano-
somes, intestinal flagellates, etc., liquid petrolatum is to be highly rec-
ommended. The chromatin staining lasts without any fading for at
least two years. The acidity of balsam causes rapid fading of the
chromatin.

Neisser's Stain for Diphtheria Bacilli
SOLUTION No. i SOLUTION No. 2
Methylene blue. ... o. i gram. Bismark brown

0.2

Alcohol 2 c.c.

Glacial acetic acid. . 5 c.c.

Distilled water 95 c.c.

Dissolve the methylene blue
in the alcohol and add it to
the acetic acid water mixture.
Filter.

Water (boiling) 100 c.c.

Dissolve the stain in the boil-
ing water and filter.

DIPHTHERIA STAINING 43

To stain: Fix the preparation. Pour on the dilute acetic acid methylene blue
solution and allow to act from thirty to sixty seconds. Wash. Then pour on the
Bismark-brown solution, and after thirty seconds wash off with water. Dry and
mount. The bodies of the bacilli are brown with dark blue dots at either end.

Neisser recommends only five seconds as the time of application of each solution.
He also recommends that the culture be only nine to eighteen hours old and that the
temperature of the incubator shall not exceed 36°C. Incubation at 37°C. gives
satisfactory results.

Ponder's Stain for Diphtheria Bacilli

Toluidin blue (Grubler) o. 02 gram.

Glacial acetic acid i c.c.

Absolute alcohol 2 c.c.

Distilled water to 100 c.c.

The film is made on a cover-glass and fixed in the usual way. A small quantity
of the stain is spread on the film and the cover-glass is turned over and mounted as
a hanging-drop preparation. The metachromatic granules of the diphtheria bacilli
stain with striking intensity. With diphtheroids, the more intense staining sharply
differentiates from ordinary cocci and bacilli, which show in the preparation only as
faint light blue bodies. It is a most excellent stain for bringing out the ascopores of
yeasts. In my opinion the stain is more valuable than the Neisser method.

Capsule Staining. — The best method for studying bacteria, as to
presence of capsules, is in the hanging drop, with the greater part of the
light shut off by the diaphragm.

In material where capsules are well developed, as in pneumonic sputum, the
Gram method of staining brings out the capsule perfectly. This is of diagnostic
value, as the more or less nonpathogenic pneumococci common about the mouth
do not seem to show a capsule when stained in this way. The India ink method of
staining gives good results for capsules.

The most beautiful method of staining capsules is the latest one pro-
posed by Muir.

1. Prepare thin film, dry and stain in carbol fuchsin one-half minute; the prepa-
ration being gently heated (steamed).

2. Wash slightly in 95% alcohol, then wash well afterward in water.

3. Flood preparation in mordant for five to ten seconds.

Mordant. — Sat. aqueous sol. mercuric chloride 2 parts.

Tannic acid (20% aqueous sol.) 2 parts.

Sat. aqueous sol. potash alum 5 parts.

4. Wash in water thoroughly.

5. Treat with 95% alcohol for one minute. (The preparation should have a pale
red color.)

6. Wash well in water.

7. Counterstain with methylene blue one-half minute.

44 STAINING METHODS

8. Dehydrate in alcohol. Clear in xylol and mount. (May simply dry specimen
with filter-paper.)

Rosenow'a Capsule Stain. — Make a very thin smear of the pathological material
and when nearly dry cover the preparation for ten to twenty seconds with 10%
tannic acid solution. Wash in water and blot. Stain with aniline gentian violet
by gently steaming for one-half to one minute. Wash in water. Apply Gram's
iodine solution for one-half to one minute. Decolorize in 95% alcohol and then
stain with alcoholic solution of eosin. Wash in water, dry and mount.

Flagella Staining. — Inoculate a tube of sterile water (gently) in
upper part, with just enough of an eighteen to twenty-four-hour-old
agar culture to produce faint turbidity. Incubate for two hours at
37°C. From the upper part of culture take a loopful and deposit it on
a cover-glass. Dry in thermostat for one to five hours or over night.
Use perfectly clean cover-glasses. To stain by

Muir's Modified Pitfield Method

1. Flood specimen with mordant. Steam gently one minute.

Mordant. — Tannic acid (10% aqueous solution) 10 c.c.

Sat. aq. sol. mercuric chloride 5 c.c.

Sat. aq. sol. alum 5 c.c.

Carbol fuchsin 5 c.c.

Allow precipitate to settle or centrifuge. Keeps only one week.

2. Wash well in water for two minutes.

3. Dry carefully — preferably in incubator.

4. Pour on stain. Steam gently one minute.

Stain. — Sat. aq. sol. alum 10 c.c.

Sat. ale. sol. gentian violet 2 c.c.

(May use carbol fuchsin instead of gentian violet.)
Stain only keeps two days.

5. Wash well in water. Dry and mount.

Zettnow's Flagella Staining Method

Solution I. — Dissolve 2 grams of tartar emetic in 40 c.c. water.

Solution II. — Dissolve 10 grams tannin in 200 c.c. water. To the 200 c.c. solution
II, warmed to 50 or 6o°C., add 30 c.c. of the tartar emetic solution. The turbidity
of the mordant should entirely clear up on heating. The mordant should keep for
months when a small crystal of thymol is added to it.

Next dissolve i gram silver sulphate in 250 c.c. distilled water. Of this solution
take 50 c.c. and add to it drop by drop ethylamine (this comes in a 33% solution)
until the yellowish-brown precipitate which forms at first is entirely dissolved and
the fluid is entirely clear. It requires only a few drops. The bacterial preparations
prepared as described above are floated in a little mordant contained in a Petri dish

STAINING PROTOZOA 45

which is heated over a water-bath for five to seven minutes. Take the dish contain-
ing the preparation off the water-bath and as soon as it becomes slightly opalescent
as the result of cooling remove the cover-glass preparation and wash thoroughly in
water. Then heat a few drops of the ethylamine silver solution upon the mordanted
cover preparation until it just steams and the margin appears black. Next wash
thoroughly in water and mount. This gives the most satisfactory results of any
method I have ever experimented with.

Spore Staining. —The most satisfactory spore staining method is
really the negative staining of the spore obtained when a bacterial
preparation is stained by dilute carbol fuchsin or Loffler's methylene
blue. The spore appears as a highly refractile piece of glass in a colored
frame.

The acid-fast method, as for tubercle bacilli, gives good results. The decoloriz-
ing, however, must be lightly done, otherwise the spore will lose its red stain.

M oiler's Method. — Fix films and then treat with chloroform for one or two
minutes. Wash thoroughly and treat with a 5% solution chromic acid for one
minute. Wash in water and then stain as for acid-fast organisms with carbol
fuchsin. Use a i% sulphuric acid solution instead of the 3% acid alcohol.

Agar Jelly Staining Method of H. C. Ross

Very clear i^% solution of agar is colored with Unna's polychrome methylene
blue, Giemsa's solution, thionin or Gram's solution of iodine. Very thin smears of
blood, faeces or gastric content sediment are made and either fixed lightly in the flame
or air dried. A drop of the melted colored agar solution is placed on the smeared
cover-glass and this is mounted immediately on a clean slide. The preparation is
ready for examination in about two minutes.

The Staining of Protozoa

Unless staining albuminous material it is well to add a little blood-
serum albumin fixative or white of egg to the preparation — about one
loopful to a smear. The serum or white of egg is best preserved by
the addition of 2 % chloroform and kept tightly corked.

Giemsa's Method. — Fix moist smears with a fixative made by adding i part of 95%
alcohol to 2 parts of saturated aqueous solution of bichloride of mercury. Keep in
this solution i to 12 hours. Now wash for a few seconds in water and then for about
five minutes with a dilute Lugol's solution (KI, 2 gm.; Lugol's solution, 3 c.c.; Aqua,
100 c.c.). Now wash in water and then in a 0.5% solution of sodium thiosulphate
to remove the iodine which was used to remove the mercury. Wash in water five
minutes, then stain with Giemsa's stain as used in blood-work for one to ten hours.
Wash and mount.

Vital Staining of Protozoa with Neutral Red Solution. — As a stock
solution one uses a 0.5% aqueous solution of neutral red.

46 STAINING METHODS

The drop of salt solution or water on the slide should be tinged a light violet-rose
color with a fraction of a loopful and the faeces or other material emulsified in this.

Protozoa take a rose-pink color with a distinct differentiation be-
tween endoplasm and ectoplasm.

Should the faeces be quite alkaline the neutral red will be decomposed with the
formation of bilirubin-like crystals.

The Giemsa formalin method described under Blood-work is of value in certain
cases.

Panoptic Method. — Highly to be recommended for the staining of protozoa,
whether in smears or in sections, is the Panoptic method.

1. Wright's or Leishman's stain for one minute.

2. Dilute with water and allow dilute stain to act for three to ten minutes. Wash
in water and then

3. Pour on dilute Giemsa's stain. Allow to stain from thirty minutes to twenty-
four hours. Differentiate with i : 1000 acetic acid solution until blue stain just
shows commencing diffusion into the acetic acid. Then wash in water, 95% alcohol,
absolute alcohol and treat with xylol and mount in liquid petrolatum.

With preparations other than blood smears, as sections, it is better to go from 95%
alcohol to oil of origanum, then mount.

Owing to the great value of a sharp nuclear picture in differentiating amoebae
it is of great importance to use some iron haematoxylin method. That of Weigert
is given in the appendix.

Mallory's Phosphotungstic Haematoxylin. — Fix moist smears, film surface down,
in Zenker's fluid for five to ten minutes. Wash in water, treat with Gram's solu-
tion and wash with 70% alcohol until all the yellow color is discharged. Wash in
water. Then stain with Mallory's phosphotungstic hcematoxylin for one-half hour.
Wash clear and mount. See appendix.

Mallory's Differential Stain for Amoebae. — Staining in saturated aqueous solution
thionin for from three to five minutes. Next differentiate in 2% aqueous solution
oxalic acid for one-half to one minute. Then wash in water, clear and mount.
Nuclei of amoebae are stained a brownish red.

Rosenbusch Iron Haemotoxylin Stain

Rapidly smear out with a toothpick a small particle of faeces or other material
containing protozoa and, while still moist, fix by Giemsa's method and, after getting
rid of the mercury with iodine followed by 95% alcohol, treat smears with a 3.5%
solution of iron-alum in distilled water for one-half hour or over night, then wash
thoroughly in distilled water.

Then stain from five to twenty minutes in the following haematoxylin stain: (i)
i% solution of haematoxylin in 95% alcohol. It takes at least ten days to ripen.
(2) A saturated solution of lithium carbonate. Add to 10 c.c. of the haematoxylin
solution 5 to 6 drops of the lithium carbonate one. Next wash well and differentiate
with about a i% solution of the iron alum. Again wash in water, pass through
alcohols to xylol and mount in balsam.

STAINING TREPONEMATA 47

Mallory's Iron Haematoxylin Method

I have obtained beautiful staining with this simple method. The great point in
technic is the watching of the differentiation.

Treat sections or moist smears fixed by Giemsa's method with 10% aqueous solu-
tion of ferric chloride for three to five minutes. Then drain off iron solution, blot
the section and stain for four minutes with a freshly made solution of i % haematoxylin
in water. To make this add a few small crystals to 4 or 5 c.c. water in a test-tube
and dissolve by heat. The stain deteriorates after twenty-four hours. Wash in
water. Differentiate with a Y±% aqueous solution of ferric chloride. The dif-
ferentiation is complete in from a few seconds to one or more minutes according to
depth of staining and thickness of film. Wash in water, pass through alcohols and
xylol and mount.

Spirochaete Staining Method (Fontana)

The smears must be air dried, not fixed by heat. Cover films with Huge's fluid
which is i c.c. acetic acid, 20 c.c. formalin and 100 c.c. distilled water. Flood films
several times with this fluid. Wash in water and cover with a mordant of 5% tannic
acid in i % carbolic acid solution. Heat the mordant on the slide until steam arises,
and allow the heated mordant to act about thirty seconds. Wash in water and
without drying pour on the silver stain, heat until steam arises, leave heated stain
on for thirty seconds, wash in water, blot, dry and examine with immersion objective.
The treponemata are brownish to black.

To make the silver stain make a J4% solution of silver nitrate in distilled water.
Then add drop by drop ammonia until a slight turbidity is produced; only a trace
of ammonia is required and any excess again clears up the solution and makes it
useless.

CHAPTER IV

STUDY AND IDENTIFICATION OF BACTERIA— GENERAL
CONSIDERATIONS

IN order to study bacteria it is necessary to isolate them in pure cul-
ture. This may be accomplished by taking one or more loopfuls of
the material and mixing it in a tube of melted agar or gelatin. From
this first tube one or more loopfuls are transferred to a second tube of
melted agar or gelatin, and from this a third transfer is made, thereby
giving us tubes in which the distribution of the bacteria is one or more
hundred times less in the second than in the first tube, and equally more
dilute in the third than in the second. When we pour the contents
of the tubes into Petri dishes we would have the bacterial colonies on
the first plate so thick that it would be impossible to pick up a single
colony with a platinum needle without touching an adjacent one. On
the second plate the distribution might be such that we should have
discrete, well separated colonies, material from which could be taken up
on the point of the needle or loop without touching any other colony.
If the second plate did not meet these requirements, the third would.

In clinical bacteriology we work almost entirely with organisms preferring blood-
heat temperature, hence it is necessary to use agar or blood-serum as standard media
for the obtaining of isolated colonies. Gelatin is of little value for this purpose in
medical work. In using agar it will be remembered that it solidifies at a temperature
slightly below 4o°C. and does not melt again until it is subjected to a temperature
practically that of boiling. Again, if the temperature of the media exceeds 44°C. it
may affect injuriously the organisms we wish to study. Consequently it requires
careful attention and quick work to inoculate the tubes, mix, transfer and pour into
plates within the limits of a temperature which injures the organisms, and one which
brings about the solidification of the agar.

Again, we not only have colonies developing from organisms which
have been fixed at the surface as the agar solidified in the plate, but
more numerous ones developing from bacteria caught in the depths of
the media. Therefore we have superficial and deep colonies. Except
to the person of great experience, all deep colonies look alike and there
is at times great difficulty in deciding whether a colony is deep or super-
ficial. It is in the matter of trying to obtain information from the

48

COLONY ISOLATION

49

differences in deep colonies that the greatest difficulties in the study
of bacteriology arise. By using the method of simply stroking plates
along five or six parallel lines from one side of the plate to the other
with a bent glass rod, platinum loop, or a small cotton swab, we obtain
colonies which are well separated and which are entirely superficial.

FIG. 9. — Petri agar plate. Made by spreading scrapings from the mouth over
sterilized nutrient agar; after forty-eight hours in the thermostat the light "colonies"
develop. Streaked plate. (Delafield and Prudden.)

We pour about 10 c.c. of agar, blood agar or Endo media into Petri
dishes and keep them in the refrigerator for immediate use.

Smeared or Stroked Poured Petri Plates. — The material as pus, faeces, throat

membrane, etc., should be evenly distributed in a tube of sterile water or bouillon;

the swab which was originally used for obtaining the material being then pressed

against the sides of the test-tube to express excess of fluid and then stroked gently

4

50 STUDY AND IDENTIFICATION OF BACTERIA

over successive lines on one plate. Or, if the organisms be very abundant, over a
second plate without recharging it from the inoculated tube.

According to my experience a very satisfactory method is to take a loopful from
the bouillon tube suspension of the pus or faeces and deposit the fluid in the platinum
loop on the left half of the poured plate then, without recharging the loop, we touch
the right half of the plate. Now taking a bent glass rod from a jar of 95% alcohol
we flame it and to cool the same we press the bent portion into the middle of the
plate. This also divides the surface of the plate into two portions. Then rubbing
the bent rod over the smaller amount of the material on the right side we carry it over
the entire right side. Then go to the loopful deposited on the left side with the rod
and rub it over this side. For urine, deposit i drop on one side and 5 drops on
the other. A smear from pus, sputum, urine or throat culture should always be made
first in order to get an idea as to the degree of dilution which is necessitated before
plating out. We use blood agar plates as routine ones when we culture from
throat, glands, joints, pus, blood, etc. The lack of translucency does not interfere
when we study the morphology of superficial colonies, using a hand lens. Then
the haemolytic zone of S. pyogenes or the green of S. viridans or the Pneumococcus
make such a medium indispensable. All pathogens grow well on it. For faeces
we use Endo's medium.

Esmarch's Roll Culture Tubes. — Having melted about 5 c.c. agar in a test-
tube we inoculate the melted medium at 45°C. and very quickly roll the tube in
a groove melted out of a block of ice. The agar sets on the sides of the tube and
colonies may be studied with a glass. Such tubes form a large amount of water
of condensation which aids in the study of streptococci. By inoculating as above,
heating to 8o°C., then rolling and filling the tube with liquid petrolatum we have
a simple method for anaerobes. Larger tubes with rolled media are useful for
culturing bacterial growth for vaccines, this technique giving a larger surface
than the slant.

To obtain isolated colonies on blood-serum or blood-streaked agar,
which can be touched and by transfer obtained in pure culture, we
simply smear the material on a slant of either medium. Then, without
sterilizing the loop, we smear it thoroughly over a second slant, and so
on to a third, or possibly a fourth or fifth.

Classification. — At present the classification of the bacteria is very unsatisfactory
from a scientific standpoint. The nomenclature abounds in instances where three
or four terms are used in naming a single bacterium, instead of the single generic
name and single specific one as is used in zoological nomenclature. This matter of
nomenclature is a subordinate factor in the confusion when we begin to investigate
and find that different names have been applied to apparently the same organism.

The slightest variation in morphological, locomotor, or biological
characteristics seems to be considered sufficient by many observers to
justify the description of a new species, and, of course, the giving of a
new name. Many of these names which are now retained were applied
prior to the epoch-making introduction of gelatin media by Koch (1881)

CLASSIFICATION 51

and consequently at a time when the isolation of organisms in pure
culture was a matter of extreme difficulty and uncertainty. One of the
first facts noted by the student in taking up bacteriology is the diffi-
culty in determining motility; this property should always be tested
on young cultures in bouillon. In Brownian movement there is a sort
of scintillating movement, but the bacterium does not move from that
part of the field. In current movement all the bacteria swarm in the
same direction, going very fast at times, and then more slowly. If in
great doubt, the mounting of the organisms in a 2 % solution of carbolic
acid will stop movement if it be true functional motility, while Brownian
and current movement are not interfered with. In true motility bac-
teria move in opposite and in all directions, and move away from the
place where first observed unless degenerated or dead.

At times we judge of motility by the presence of this characteristic
in a few of the organisms seen in the microscopic field, the vast majority
of the bacteria not showing motility. A source of error can be present
when the bacteria are emulsified in a drop of water which might contain
motile bacteria.

Reaction of media is a factor of the greatest importance in causing variation in
the functions of bacteria, and is one which has until recently been almost entirely
neglected. In describing an organism at the present time it is always necessary
to note the reaction of the media, the temperature at which cultivation took place,
and the age of the culture when examined.

In the following keys the term bacterium has been used as a general
designation for all schizomycetes. Migula calls motile rod-shaped
organisms bacilli, and nonmotile ones bacteria. Lehmann and Neu-
mann call spore-bearing organisms bacilli, and nonspore-bearing ones
bacteria.

The B. typhosus is very motile and does not possess spores. According to Migula,
it would be the Bacillus typhosus; according to Lehmann and Neumann, the Bac-
terium typhosum. The. B anthracis has spores and is nonmotile. Hence it would be
Bacterium anthracis, according to Migula, and Bacillus anthracis, according to
Lehmann and Neumann.

In the use of the keys at the head of each group of organisms it will be observed
that the primary separation is on the basis of morphology — the cocci in one group,
the bacilli in three subgroups: one for those rod-shaped organisms showing branch-
ing and curving forms, one for the spore bearers and one for the simple rods. The
spirilla are grouped by themselves.

An important method of differentiation is the reaction to Gram's
stain. It should be remembered that organisms carried along on arti-

STUDY AND IDENTIFICATION OF BACTERIA

CHART FOR STUDY OF BACTERIA

.Date.

Name JSource

Tbrm Arrangement. .

Size, length Mreadih Extreme length....

Capsules Spores, Central Terminal.

Motility. Pleomorphism .._;._

Staining, reaction, Loffler, Gram, CLcid fast.

Pathogenesis-.- White mouse Guinea pig. J?abbii.

AEROBIC OR FACULTATIVE

N.RGlucose L.Mannite L.Dulcite L Maltose L.Saccharose Lit.Lactose

Gelatin stab

A

Qgar slant

Cigar,
Glycerin agar

OBLIGATE ANAEROBIC

Stood agar,or Endo-or

Serum agar Lactose litmuJ3

tyar

A

Glucose agar Litmus milk Egg media, or Sterile tissut

Biocd serum, Glucose bouillon

stab

Special media:

Notes-....

FIG. io.— Bateriological Chart in use at the U. S. Naval Medical School. The
mimeographed sheets are 8 by 14 inches. Red and blue pencil shading charac-
terizes acid or alkaline reactions in sugar tubes. Outlines of colony in plate rings
are made in pencil as is also done on slant figures.

MOTILITY

53

ficial media often lose their Gram-staining characteristics; hence it is
desirable to determine this staining reaction in cultures freshly isolated.
Be sure that the stains, especially the aniline gentian violet and the
iodine solution, have not deteriorated. There is no more important
stain than this, and none which requires greater experience. The chief
causes of conflicting results are i. working with old cultures and 2. not.
having satisfactory staining solutions.

FIG. ii.— Series of stab cultures in gelatin, showing modes of growth of different
species of bacteria. (Abbott.')

Motility, as stated above, is at times difficult to determine. For this purpose
young eighteen-hour-old bouillon cultures are preferable, and the preparation should
be made by applying a vaseline ring to the slide, then putting a drop of the bouillon
culture in the center of the ring (or a drop of water inoculated from an agar slant
growth), then putting on a cover-glass. By this method current movement is done
away with and the preparation keeps for hours. This is a convenient method for
agglutination tests. The hanging drop with a concave slide is ordinarily used.
With this, cut down the light and focus on the margin of the drop with the %-
inch objective before examining with a high dry objective (% inch).

54 STUDY AND IDENTIFICATION OF BACTERIA

Liquefaction of gelatin is a very important means of differentiating. When room-
temperature incubator is not at hand (20° to 22°C.), it is better to put the inocu-
lated gelatin tube in the body-temperature incubator, and from day to day test the
power of solidifying with ice- water. If the organism digests the gelatin (a liquefier),
the medium will remain fluid when placed in ice- water; if the organism is a non-
liquefier, the medium in the tube becomes solid. Of course we lose the information
to be obtained from the shape of the area of liquefaction.

For routine work the only sugar media used are glucose and lactose
bouillon. These are of the utmost importance in differentiating organ-
isms of the typhoid and colon group. Following Ford, these intestinal
bacteria have primarily been separated by their action on litmus milk
— whether turning it pink or only slightly changing or not changing at
all the original color.

Colony Isolation. — Examine the colonies on Petri plate at first with the unaided
eye, then with a hand magnifying glass or low-power objective, using reflected and
transmitted light alternately. Having determined the presence of two or more dif-
ferent kinds of colonies, make a ring with wax pencil around one or more of each
kind of colony, numbering them. The slides or culture tubes used in determining
the species of organism present in the plate should bear the same number as that
of the colony from which the material was taken. A convenient procedure is to
put a loopful of water on a clean coverglass and emulsify material from a colony
in it. Then invert over a concave slide without vaselining the circumference of
the concavity. After examining for motility, smear out and dry the bacterial
preparation. Then fix in the flame and stain with aniline gentian violet for two
to five minutes. Wash and mount the preparation in water. Afterward pass
through the usual Gram technic.

After this inoculate the various culture media from similar colonies. One may
inoculate a tube of bouillon from a single colony and later on inoculate the other culture
tubes.

In testing for gas production it is better to use the Durham fermenta-
tion tube as small amounts of gas may not be easily detected with deep
stab cultures into glucose or lactose agar.

If a Durham or Smith tube, or a slant of Russell's double sugar medium be not at
hand the production of gas may be determined by observing bubble formation on the
surface of the sugar bouillon culture. As none of the pathogenic cocci produce gas,
fermentation tubes are unnecessary where cocci are to be studied. The litmus milk
tube gives data as to acid production.

An important point is to wait at least forty-eight hours (in the case
of M . melitensis, four to seven days) before reporting on the cultural
findings on the agar, blood agar or blood-serum slant or plate upon
which the material is smeared (pus, exudate, blood, etc.).

Indol production is of but slight aid in differentiating organisms.

55

Anaerobiasis. — If it were not for the fact that we have so many facultative anaerobes
(organisms growing under anaerobic as well as aerobic conditions) it would be of
practical utility to make this biological variation our first step in the study of an
unidentified organism. At any rate it is well to remember that the causative organ-
isms of plajue, tuberculosisfmfluenza, gonorrjioea. pneumococcal pneumonia and
glanders are obligate aerobes ^/vhile those oTftetanus, botulism, gasjfangrene and
malignant oedema are obligateanaerobesj The p^ogenic cocci as well as the causa-
tive organisms of choleraTtyprioid, parathyphoid and anthrax are facultative anae-
robes; they are, however, always studied under aerobic conditions. The colon
bacillus as well as organisms of the Friedlander group are also facultative anaerobes.

Should an organism be encountered in original investigations these
requirements as to etiological relationship should be carried out (Koch's
postulates) :

i. The organism should be constantly present in that particular pathological
condition. 2. Such bacteria should be isolated in pure culture from the pathologi-
cal material. 3. Such pure cultures when inoculated into suitable animals should
reproduce the pathological conditions and should be capable of a second isolation in
pure culture from such an experimental animal. For various reasons, such as unsuit-
able animals or artificial media, these requirements are impossible of execution with
several organisms which are generally recognized as the causes of certain diseases.

ANIMAL EXPERIMENTATION

The experimental animals most frequently employed in the diagno-
sis of bacterial diseases are the guinea-pig, the rabbit, the white rat
and the white mouse. In the following diseases the most suitable ani-
mals for inoculation are:

1. Tetanus — mice or guinea-pigs, subcutaneously. The spasms begin in the
limbs nearest the site of inoculation.

2. Pneumococci and streptococci — mice, intraperitoneally, or rabbits, intraven-
ously.

3. Staphylococci — rabbits.

4. Diphtheria, tuberculosis, anthrax and malignant cedema— the guinea-pig,
subcutaneously.

5. Glanders and cholera— the guinea-pig, intraperitoneally.

6. Plague— guinea-pigs, cutaneously or subcutaneously.

In the cutaneous method of infection the material, as from a plague bubo, or the
sputum from pneumonic plague, is thoroughly rubbed with a glass rod upon the
shaven surface of the guinea-pig.

In the subcutaneous method one can use a hypodermic needle (the all-glass
syringe with platino-iridium needle is the best) or an opening can be cut with the
scissors, a pocket then opened up with the forceps and a piece of tissue inserted to
the bottom of the pocket with the forceps.

One can seal the incision with collodion.

56 STUDY AND IDENTIFICATION OF BACTERIA

The large ear vein of the rabbit is used for intravenous inoculation. This can be
made to stand out with either hot water or xylol.

In intraperitoneal injections the animal is best held head down so that the intes-
tines gravitate downward. The shaven skin is pinched up and the needle inserted in
the median line.

In certain cases infection is secured by feeding the material to the experimental
animal. The material may be mixed with the food or introduced into the stomach
by a tube.

CULIURING MATERIAL OBTAINED AT BIOPSY OR AUTOPSY OF MAN OR

ANIMALS

It is customary to sear with a heated knife or spatula a spot on the
surface of the organ from which cultures are to be made. Then intro-
duce at this point a sterile platinum spud and inoculate tubes of culture
media. The platinum loop may be used where an incision is made
into the organ with a sterile knife. The ordinary platinum loop, how-
ever, bends too easily.

A bacteriological capillary pipette is a good instrument for taking up material.
After some practice one can do good work with a rubber bulb capillary pipette,
especially in taking up blood from right heart or blood-vessels. When great pre-
caution is necessary, as in culturing a removed gland or organ, the piece of tissue
may be dropped into 5% formalin solution for a few minutes, then washed in sterile
salt solution; next placed in a sterile Petri dish and the material obtained from the
center. It may be dropped for a few seconds into boiling water to sterilize the
surface. When autopsying experimental animals it is well to dip the dead animal
into 3% tricresol solution before opening up the body.

CHAPTER V

STUDY AND IDENTIFICATION OF BACTERIA— COCCI. KEY

AND NOTES

Streptococcus Forms. — Cells divide to form chains.
I. Gelatin not liquefied.

1. Haemolytic zone on blood agar.

(a) Very slight acidity in lactose litmus bouillon. S. pyogenes. Tends to
produce arthritis in experimental animals. Often a granular sediment in
bouillon.

(&) Marked acidity but no gas production in jactose litmus bouillon. S.
lacticus. Nonpathogenic. Forms diffuse cloudiness in bouillon.

2. G<re^nish appearance about colonies on blood agar.

(a) No tendency to capsule formation. S. viridans. Produces endocarditis
*• • in experimental animals.

(&) Distinct capsule formation in pathological material or on favorable media.
S. lanceolatus (Pneumococcus). Gram-positive, lance-shaped cocci with
bases apposed within a capsule.

(c) Very marked capsule development on all media. S. mucosus. A. strepto-
coccus with extraordinary capsule development, up to loju in width, S.
mesenterioides, is not pathogenic.
II. Gelatin liquefied.

Streptococcus coli gracilis. (Cocci quite small — 0.2 to 0.4/1. In faeces.)

A tube-like liquefaction; chains rather long; only slight growth on agar.

Constant inhabitant of stools of meat diet.
Sarcina Forms. — Cells divide in three dimensions of space. (Packets).

A. No pigment production on agar.

(a) Sarcina alba. (Colonies finely granular.)

(b) Sarcina pulmonum.

B. Yellowish pigment.

(a) Sarcina lutea. (Colonies coarsely granular.)
(b)\ kSarcina flava. (Colonies finely granular.)

C. Rose-red pigment.
(a) Sarcina rosea.

Micrococcus Forms. — Cells divide irregularly in various directions.
I. Gram-positive cocci.
A. Cocci-round.

i. Divide in two planes at right angles. Tetrad formation. Merismopedia.
(a) M. tetragenus. Mo'ist white viscid colonies. No liquefaction of gela-
tin. Capsule.

57

58 STUDY AND IDENTIFICATION OF BACTERIA

2. Divide irregularly. Bunch of grapes arrangement. (Staphylococci.)
(fl) Gelatin not liquefied. M. cereus albus.

(6) Gelatin liquefied. ( "' (Staphytococcu.) pyogenes albus.

( M. (Staphylococcus) pyogenes aureus.
(c) Gelatin very slightly liquefied.

S. epidermidis albus. (Stitch coccus.)
B. Cocci — biscuit-shape.

Diplococcus crassus. (May be mistaken for Meningococcus.)
On ordinary agar we have a scanty growth resembling the Streptococcus. Colo-
nies on ascites agar are smaller than those of Meningococcus. It produces acid in
glucose, maltose, lactose and saccharose.
II. Gram-negative cocci.

only at about incubator temperature.

1. Grow only on blood or serum media. Gonococcus.

2. Grow on blood-serum media, or glycerine agar.

(a) Diplococcus intracellularis_rneningitidis. Produces acid in glucose

and maltose but not in lactose nor saccharose.
\^. Grows on ordinary media. Micrococcusjnelitensis.
B. will grow at room temperature as well as at 37°C.

(a) Micrococcus catarrhalis. Does noj: produce acid in glucose, maltose,
lactose nor saccharose. ^

(b) M. pharyngis siccus. Colonies dry and tough and adhere to medium.
NOTE. — Other biscuit-shaped Gram-negative organisms resembling the Meningo-
coccus are (a) Diplococcus flavus. The colonies show yellow pigment and we have
three varieties according to the depth of the yellow color, (b} M. pharyngis siccus,
with yellowish, dry tenacious colonies and (c) M. cinereus with coarse dry colonies
on ascitic agar. Like M . catarrhalis, it does not ferment any of the above-mentioned
sugars. The individual cocci, however, are larger and more oblong in shape.

STREPTOCOCCUS FORMS

Those cocci tending to arrange themselves in chains are usually
described as streptococci. (Ogston, 1881; Rosenbach, 1884.)

When we consider that certain bacilli at times assume an arrange-
ment which we term streptobacilli, yet have no relationship, it would
suggest that the matter of chain morphology is simply a characteristic
common to many entirely different cocci.

Again old laboratory cultures of streptococci may show alternations of cocci and
rods giving the appearance of the dots and dashes of the Morse code. Furthermore
unsuitable media may bring about various involution types in an organism pri-
marily streptococcal.

It is often difficult to distinguish streptobacilli from streptococci morphologically
and the same is true of diplococci and diplobacilli. These bacillary pairs and chains,
however, often show bipolar staining and are almost invariably Gram-negative.

While streptococci tend to assume chain formation in pus and tissues they often
appear as diplococci in blood.

VIRULENCE IN STREPTOCOCCI 59

The essential point to bear in mind is that the finding of a strepto-
coccus does not necessarily explain an infection, because normally
streptococci are among the organisms most frequently and abundantly
found in plates made from normal buccal and nasal secretions. It is
well to be very conservative When reporting streptococci as the etiolog-
ical factor from lesions of the throat or nose.

Probably the most practical point in the differentiation of streptococci, next to
that of pathogenicity, is the occurrence of long or short chains, the virulent ones
tending to appear in chains of from 10 to 20 cocci, while the normal inhabitants of
the nose, mouth and faeces generally tend to be in shorter chains.

As regards virulence, this is exceedingly variable — it is soon lost, but
may be restored either by inoculating streptococci along with various

FIG. 12. — Streptococcus pyo genes, (Kolle and W assermann.)

other organisms or by passage through successive rabbits. The rabbit
is the most susceptible animal and should be inoculated in one of the
prominent ear veins. If the needle of the syringe is not inserted in
the vein it will be difficult to force in the material and a swelling will
immediately show itself.

Recently isolated cultures from human infections are not very virulent for animals.
Passage through the rabbit, however, enormously increases the virulence. It may
be more convenient to inoculate a mouse at the root of the tail. If the culture is
very virulent, it becomes generalized and death occurs in two or three days. If less
virulent, a local abscess forms.

Besides the morphological and pathogenic variations, Schottmuller
has noted differences where these organisms are grown on i part of

60 STUDY AND IDENTIFICATION OF BACTERIA

blood and 3 to 6 parts of agar. On this medium Strep, erysipdatis has
a hemolytic action, the laking of the red cells bringing about a more
or less clear ring surrounding the colony. This organism is often termed
S. hcemolyticus. It tends to produce suppurative arthritis in rabbits
while S. viridans causes endocarditis and the Pneumococcus an acute
sepsis.

The haemolytic substance is much like a ferment and is found in filtrates of cul-
tures. The short-chain streptococci do not have a haemolytic halo. They also
have a greenish appearance like the Pneumococcus (S. viridans}. The Pneumococcus
has a greenish zone. Streptococci which are profoundly toxic and which have been
isolated from milk-borne epidemic sore throats differ from the ordinary S. pyogenes
in being encapsulated, not tending to form chains and producing only slight haemoly-
sis on blood agar.

Some of the English authorities have introduced biochemical meth-
ods of differentiating: the Strep, pyogenes coagulating milk, reducing
neutral red, and producing acid in lactose, saccharose and mannite
media.

S. pyogenes does not produce acid in inulin media while the Pneumococcus does.

The Pneumococcus and Streptococcus mucosus ferment inulin and give greenish
colonies. Of streptococci not fermenting inulin we have S. pyogenes, with its haemo-
lytic zone around the colony; S. viridans, with its green colony, and S. f&calis, which
gives colonies neither distinctly green nor haemolytic.

A freshly prepared solution of sodium taurocholate, 5%, added to an equal
amount of a twenty-four-hour bouillon culture of S. pyogenes does not disintegrate
the cocci or, at any rate, not within a few minutes. The reverse is true of the
Pneumococcus.

( iWhen we consider the biochemical variations which a single organism, as the
colon bacillus, may exhibit, the value of such methods of differentiating may well be
questioned. The question of the symbiotic relationship, which, when established
between two or more bacteria, may cause harmless organisms to take on virulence,
would appear to be a more important consideration.

Almost without exception, human streptococci are Gram-positive.
Their colonies are quite small but distinct and discrete. In^appear-
ance the colonies of streptococci and pneumococci are practically iden-
tical. In a blood-serum throat culture Pneumococcus and Streptococcus
colonies are the smallest, diphtheria ones are quite small and discrete,
but slightly flatter. (Always examine the water of condensation for
streptococci.) The Sarcina and Staphylococcus colonies are much
larger.

Streptococcic colonies on blood agar are much more moist and luxuriant than on
ordinary agar. A very important point, in judging whether a Streptococcus or

GASTRIC ULCER AND STREPTOCOCCI 6 1

other organism is pathogenic in a given infection, is to examine smears from the pus
or other material in a Gram-stained specimen for information as to abundance and,
in particular, phagocytosis of any organism, before plating out.

Streptococci are commonly the cause of diffuse phlegmonous inflam-
mations, while the staphylococci cause circumscribed lesions. Strepto-
cocci cause necrosis and do not characteristically produce pus. The
importance of the Streptococcus as a secondary infection in diphtheria,
tuberculosis, small-pox, and even in typhoid fever must always be kept
in mind. It is this infection which does not respond to diphtheria
antitoxin, and not the diphtheria one.

Rosenow has reported on the rather constant presence of streptococci in gastric
and duodenal ulcers removed at operation, under which circumstance the number and
variety of bacteria present are comparatively few. The strains from 27 chronic
ulcers gave grayish-green colonies on blood plate, were in short chains and diplococci,
produced much acid and turbidity in dextrose broth and showed a low-grade virulence.
When injected into dogs, rabbits and guinea-pigs they showed a tendency to localize
in the mucosa of stomach and duodenum, causing ulceration in a large percentage
of cases.

Streptococci as well as colon infections are always to be thought of in connection
with cholecystitis and appendicitis.

It has been claimed that scarlet fever is a streptococcal infection (S. anginosus).
Klimenko found streptococci only n times in the blood of 523 cases of scarlet
fever. The Dohle inclusion bodies of the disease suggest chlamydozoal virus.
Mallory has very recently claimed that scarlet fever is due to a diphtheria-like
bacillus. It is found in the same locations as the diphtheria organism and also
produces a toxin which, however, is less virulent and only produces inconspicuous
lesions. The membrane formation in the throat in scarlet fever is due to streptococci.

When freshly isolated from human lesions streptococci often show only a slight
virulence for animals. Hence massive doses are indicated and intravenous or intra-
peritoneal injections. The guinea-pig is not very susceptible to streptococci; the
rabbit and white mouse being the animals of choice.

In nondiphtheritic anginas, puerperal fever, ulcerative endocarditis
and coccal enteritis it is the Streptococcus which is usually the cause.
It has been claimed that acute articular rheumatism is due to a short-
chain streptococcus (M. rheumaticus), which is best isolated from
material from an acute joint infection, but may also be isolated occa-
sionally from the blood. It produces much acid and clots milk in two
days. The growth is described as being more luxuriant than that of
S. pyogenes. It is about 0.5^ in diameter.

The majority of investigators have reported streptococci from acute joint inflam-
mations and bacilli from chronic infectious joint affections. Goadby has considered
a streptobacillus, somewhat resembling Ducrey's bacillus of chancroid, which ex-

62 STUDY AND IDENTIFICATION OF BACTERIA

hibits marked pleomorphism and Gram variations, and grows best on egg albumin
agar of plus 3 reaction as the cause of arthritis deformans and alveolar osteitis.
Inoculation of cultures of this organism into or around the knee-joints of rabbits
has produced lesions similar to those of rheumatoid arthritis.

Of the nonpathogenic streptococci the most important one is S. lacticus, which
is described under milk. This differs from S. pyogenes in growing at lower tempera-
tures and having greater viability. It is a normal inhabitant of cows' dung.

SARCINA FORMS

These are best observed in hanging-drop preparations, when they
can be seen as little cubes, like a parcel tied with a string, and by noting
them when turning over, it will be seen that they are different from the
tetrads which only divide in two directions of space. At times the
packet formation is not perfect and it will be difficult to distinguish
such as sarcinae. All sarcinae stain by Gram. If the staining of sar-
cinae be too deep it may obscure the lines of cleavage. Sarcinae are non-
motile.

i ^Various sarcinae have been isolated from the stomach, especially when there is
stagnation of stomach contents. Sarcinse have also been found in the intestines.
In plates the S. lutea is frequently a contaminating organism, being rather constantly
present in the air. The demonstration of sarcina morphology should always be
made from liquid media, as bouillon. Urine makes an excellent medium.

MICROCOCCUS FORMS

This grouping includes all cocci which do not show chain or packet
formation. It will be found convenient to divide them into two classes
according to their staining by Gram. The M. tetragenus, S. pyogenes
aureus and the Pneumococcus stain by Gram, while the Gonococcus,
the Meningococcus, the M . catarrhalis and the M . melitensis are Gram-
negative.

M. Tetragenus. — This organism is frequently found associated with
other organisms in sputum, especially with tubercle and influenza
bacilli. The colonies are white, slightly smaller than staphylococci and
are quite viscid.

It was formerly considered unimportant in disease, but the idea now prevails that
it is responsible for many abscesses about the mouth, especially in connection with
the teeth. Injected subcutaneously into Japanese mice, it produces a septicaemia
and death in three or four days. The blood shows great numbers of encapsulated
tetrads. It has been reported twice as a cause of septicaemia in man.

STAPHYLOCOCCI

Staphylococci. — To cocci dividing irregularly and usually forming
masses which are likened to clusters of grapes the term Staphylococcus
is applied. While there have been experiments which show that by
selecting pale portions of a yellow colony, eventually a white colony
could be produced, yet, as a practical consideration, it is convenient
to consider at least two types of Staphylococci: the Staphylococcus pyo-
genes aureus and the Staphylococcus pyogenes albus.
In culturing from the pus of an abscess or furuncle
we generally obtain a golden coccus, while in ma-
terial from the nose or mouth, the Staphylococcus
colonies are almost invariably white. As regards
the common skin coccus, this will be found to pro-
duce a white colony. A coccus which very slowly
liquefies gelatin and has been supposed to cause
stitch abscesses is the 6*. epidermidis albus.

While it is customary to look for a golden colony in the
case of organisms showing virulence, yet at times a cream-
white colony may develop from cocci of great virulence.
Staphylococci show marked resistance to dessication and
dried pus may contain live organisms for months. Old
bouillon cultures of Staphylococci contain a ferment-like
substance, leucocidin, which disintegrates leukocytes. Such
cultures may also show a haemolysin and when filtered and
injected into animals show destructive action on cells of
various organs. Amyloid change may be caused in animals
by repeated injections of either living or dead cultures.

The S. pyogenes citreus is considered as of very feeble
pathogenic power. Certain cocci whose colonies have pre-
sented a waxy appearance have been designated as S.
cereus albus and S. cereus flavus, respectively. They are of
very little practical importance. The Staphylococcus pyogenes JjJJJJ? ^/teeT old!
aureus grows readily at room temperature, but better at 37°C. (Mac Neal.)
It coagulates milk and renders bouillon uniformly turbid.

It grows on all media, as blood-serum, agar, potato, etc. It has been proposed to
distinguish it from skin Staphylococci by its power of producing acid in mannite.
Ordinarily the individual cocci are about IJJL in diameter, but they vary greatly in
size according to the age of the culture and other conditions. The "aureus/' as it
is frequently called, is not only often found in circumscribed processes, but it is a
frequent cause of septicaemia, osteomyelitis, endocarditis, etc. In the tropics
staphylococcal infections often show great virulence and clinically may resemble
streptococcal ones. Smears from such erysipelatoid lesions show diplococcal
morphology, often phagocytized. A pemphigoid eruption in children is often
staphylococcal (Pyosis).

In infection of bone tissue the Staphylococcus is by far the most frequent cause.

FIG. 13. — Gelatine

04 STUDY AND IDENTIFICATION OF BACTERIA

It is well to remember that insignificant staphylococcal infection may lead to sep-
ticaemia. In the tropics, where resistance is often lowered and staphylococcal skin
infections common, continued fevers are often septicaemias. It is the organism
most frequently concerned in terminal infections. The lowered resistance of the
patient permits of their passage through barriers ordinarily resistant. Not only
should this be kept in mind when such organisms are isolated at an autopsy, but
as well the fact that their entrance may have been agonal or subsequent to death.
Vaccines have been most successful in the treatment of staphylococcal infections.
They stimulate phagocytosis. Pyelitis may be due to staphylococci.

The Pneumococcus of Fraenkel. — (Pasteur and Sternberg in 1880.
Fraenkel, 1884, isolated it from normal persons as well as pneumonia
patients. Inoculated mice and rabbits. Hence FraenkePs organism.

FIG. 14. — Pneumococcus, showing capsule, from pleuritic fluid of infected rabbit,
stained by second method of Hiss. (Mac Neal.}

Weichselbaum accurately differentiated organisms causing pneumonia
in 1886.) This is by far the most common cause of pneumonia, whether
it be of the croupous, catarrhal, or septic type. It is also frequently
found in meningitis, empyema, endocarditis and otitis media. It
should not be confused with the pneumobacillus of Friedlander, which,
although possessing a capsule like the Pneumococcus, differs from it by
being Gram-negative, being a bacillus and having large viscid colonies.
The Pneumococcus is the cause of more than 80% of the cases of
pneumonia.

It does not grow below 2o°C. and is best cultivated on blood-serum, or blood-
streaked agar. On plain agar it grows as a very small dew-drop-like colony, which

THE PNEUMOCOCCUS 65

is slightly grayish by reflected light. It produces considerable acid, thus acidify-
ing and usually coagulating litmus milk. It produces acid inynulin media which
the Streptococcus fails to do. The colony is smaller and more transparent than a
Streptococcus colony. In sputum or other pathological material it is best recognized
by the presence of a capsule inclosed in which are two lance-shaped cocci with their
bases apposed. In artificial culture we rarely get the capsule. It also sometimes
grows in short chains like a Streptococcus. The best medium for differentiating is
the serum of a young rabbit; in this it grows as a diplococcus, while streptococci
show chains. Kosenow has by combining passage in animals with culturing sym-
biotically with B. subtilis changed the Pneumococcus into a haemolytic Streptococcus.
The best method of isolating it in pure culture is to inject the sputum into the
marginal ear vein of a rabbit or subcutaneously into a mouse. Death results from
septicaemia in about two days and the blood teems with pneumococci. Usually
the Pneumococcus quickly loses its virulence, and also dies out in a few days unless
transferred to fresh media. The best medium for its preservation is rabbit's blood
agar; this also maintains the virulence. On this medium the colonies are larger
than on agar and they present a greenish appearance.

The Pneumococcus growth emulsifies very readily and evenly so that
suspensions for vaccines are easily made.

It is a well-known fact that Pneumococcus is a frequent inhabitant of the nasal,
pharyngeal. and buccal cavities. The explanation of infection is either on the
ground of lowered resistance of the patient or enhanced virulence of the organism.
Results from the use of antipneumococcic sera have been of only slight value. Such
sjira^are useless against infections with_other strains. Vaccines appear to be value- >1-
less in pneumonia but may be useful in local infections. They probably stimulate
opsonin production. Oscar Richardson has reported an organism in cases of
lobar pneumonia, cerebrospinal meningitis, mastoid disease, etc., bearing resemb-
lance to both pneumococci and streptococci — the Streptococcus capsulatus. Other
names are Streptococcus mucosus and Pneumococcus mucosus. It is very virulent
for mice but less so for rabbits. Park isolated it twice from 20 cases of pneu-
monia. It differs from the Pneumococcus in that the colonies on blood-serum are
viscid and like irregular flecks of mucus. The characteristic culture is a glucose
agar stab. (Reaction must not exceed +0.5.) From the line of puncture there are
flail-like projections extending outward from ^ to 24 inch. The capsule persists
on ordinary culture media. This organism resembles the Streptococcus of Bonome
of the French.

In a study of blood and sputum cultures from 32 cases of lobar pneu*
monia Hastings and Boehm found blood and sputum positive bacteriologically
in ii cases. In nine of these cases the Pneumococcus was isolated and in 2
a haemolysing Streptococcus. In the other 21 cases the sputum cultures
were bacteriologically positive in 18 of the cases and negative in 3. In
9 cases the Pneumococcus was isolated, in 2 cases B. coli, in i case M. catar-
rhalis, in i case a Staphylococcus, in 2 cases staphylococci and streptococci,
in i case B. influenza. The percentage of positive blood cultures was 30.3.
Cole obtained 30% of positive blood cultures. The blood was taken into flasks of
bouillon in dilution of 1-50.
5

66

STUDY AND IDENTIFICATION OF BACTERIA

Diplococcus Crassus. — This is a Gram-positive, kidney-shaped dip-
lococcus, which might be confused with the M. catarrhalis or the Menin-
gococcus by ordinary staining methods. It is larger than the Meningo-
coccus.

It is not strongly Gram-positive as one may find examples in the same prepara-
tion about which doubt may be entertained. It ferments lactose and saccharose
as well as glucose and maltose.

In throat cultures I have isolated on several occasions a Gram-positive diplococcus
which is at times biscuit-shaped, at times irregularly spherical. It possesses two
or three metachromatic granules, so that in a Neisser stain for diphtheria the
appearance of these granules may be confusing.

Using Ponder's toluidin blue stain I have observed granule staining in organisms
of round or oval morphology which were suggestive of the ascospore staining of
yeasts. Staphylococci may show granules with Ponder's stain.

Gram-negative Cocci. — It is important to bear in mind that there
are many cocci of varying shapes, which in cultures or in smears from

FIG. 15. — Gonococcus. Film from urethral pus. (Coplin.)

the throat, nose or faeces are Gram-negative. These are not well classi-
fied or described. To distinguish the three important kidney-shaped
diplococci, it can be most easily accomplished by cultural methods,
using hydrocele agar (ascites or blood agar will answer), ordinary blood-
serum and plain agar. The Gonococcus will only grow on the hydro-
cele agar; the Meningococcus will grow on this, but likewise grows on

GONOCOCCUS 67

ordinary blood-serum. The M . catarrhalis will grow on plain agar as
well as on other media.

Other Gram-negative organisms of confusing morphology are M . pharyngis siccus,
the colonies of which show great crinkly dryness, and M. pharyngis flavus.

Gonococcus (Neisser, 1879). — This organism is characteristically a
diplococcus, the separate cocci being plano-convex with their plane
surfaces apposed. (Biscuit shape, coffee-bean shape.) They are gen-
erally found grouped in masses of several pairs, most strikingly in pus
cells or epithelial cells, but also found extracellularly. Except in the
height of the disease, there is a great tendency for the organisms to
show involution forms, so that instead of biscuit-shaped diplococci we
have round, irregular and uneven cocci.

It is therefore advisable in searching smears from chronic gonorrhoea to continue
the search of Gram-stained specimens until some fairly typical diplococci are found.
There is nothing requiring greater discrimination than a diagnosis from such a ,

smear. At the commencement of a gonorrhoea the epithelial cells are abundant
and gonococci are found, adhering to them or lying free. Later on, at theacjmejpf ^r
the discharge (the creamy, abundant discharge), it isjn the piis^cells wj^md them
and they may be so abundant that 10 to 20% of the pus cells may contain theni.
In the subacute_stage the epjthelial .cells, which practically disappear when the
discharge is so abundant, begin tojreappear, and in the chronic stage the epithelial
cells_are the chief ones, and are the ones on which we find an occasional gonococcus,
often distorted in shape. In gonorrhceal ophthalmia the gonococci may show
appearances in the conjunctival epithelium resembling the inclusion bodies of
trachoma.

The best method of diagnosis in cases of chronic gonorrhoea is to have the patient
eat the stimulating food previously interdicted, to take active exercise and to have
a sound passed. To obtain material for examination the glans penis should be
washed and the patient who has presented himself with a full bladder should pass
a portion of the contained urine. Next the prostate and seminal vesicles should be
massaged with the patient standing but bent over and the penis pendant. The
drops of discharge from the massage should be received in a small Petri dish and
finally the remaining urine should be passed into a sterile bottle. Smears and
cultures should be made from the sediment of the two urinary specimens and
from the secretions of the massaged prostate and vesicles.

The smears made from the resulting discharge or centrifuged urine will probably
contain gonococci if they are present in the urethra. In the female the favorite
sites are the urethra and the cervix uteri. In municipal examinations it is customary
to make two smears: one from the urethral meatus and a second from the cervix.
The vagina is not a suitable soil for their development. In female children it is
most often found in the discharge of the vulvovaginitis. Gram-stained smears from
pus sediments of urine, especially in pyelitis or cystitis, may show coccoid forms of
B. coli, which may be phagocytized and thus be reported as gonococci.

68 STUDY AND IDENTIFICATION OF BACTERIA

In addition to the genital organs, the Gonococcus may at times invade and be
isolated from the eye (gonorrhceal ophthalmia), the joints, rarely as a cause of
endocarditis and possibly as the factor in septicaemia. Grown upon hydrocele or
ascites agar, or blood-streaked agar, or upon blood agar from man or the rabbit,
the colonies appear as irregular, minute, dew-droryipots. By the second or third
day the involution forms are abundant, and within four to seven days the culture
will probably be found to be dead. Unless frequent transfers are made, it will be
best kept alive on blood agar. The organism grows best at 37°C., and will not
grow below 25°C. It will not grow on plain or glycerine agar or ordinary blood-
serum unless the transfer of considerable pus in inoculating the slants gives it a
suitable culture medium. In material from joints, it is in the fibrin flakes that the
gonococci are most apt to be found, if found at all.

Animals do not contract gonorrhoea. Even in monkeys urethral in-
oculations of gonococci are negative. The organism is killed in five
hours by a temperature of 45°C. and speedily by drying. In moist
smears of pus it may live for one or two days.

By heating the blood-streaked agar tubes to s6°C. for twenty minutes (inactiva-
tion-destroying complement and hence bactericidal power of blood on slaflt) greater
success in primary cultures will be obtained.

In culturing gonococci the transfer of material to culture media
should be made with the least delay possible.

The most satisfactory medium is Thalman's medium upon the slanting surface
of which we have deposited two or three drops of human serum. Blood may be
taken from a vein or the Wright U tube may be used and after centrifuging the
sterile serum is taken off with a capillary bulb pipette and deposited on and smeared
out on the slant. We now use Vedder's starch medium.

Diplococcus Intracellularis Meningitidis (Weichselbaum, 1887).—
This is the organism of epidemic cerebrospinal meningitis, and is fre-
quently termed the Meningococcus. The diplococcus is Gram-negative
and biscuit-shaped and is, like the Gonococcus, chiefly contained in pus
cells. It is also found free in the cerebrospinal fluid withdrawn from
cerebrospinal fever cases. There is a greater tendency to variation
in size and shape than is the case with the Gonococcus, which latter, in
fresh material, shows a striking uniformity morphologically. The
Meningococcus is at times not abundant. Early in the case, however,
the picture may be similar to that of gonorrhceal smears.

On blood-serum the colonies appear after twenty-four to forty-eight hours as
discrete, very slightly hazy colonies, about Ho mch in diameter. On serum
agar, as ascites or hydrocele agar, they grow best and show as faint bluish
colonies about i to 2 mm. in diameter. They are larger than Streptococcusjzi
Pneumococcus colonies. Unless^ considerable cerebrospinal fluid is transferred

THE MENINGOCOCCUS 69

with the inoculating loop, they do not grow on plain agar. They will grow at times
on glycerine agar. The organism is very sensitive to light, cold and drying. It
ferments glucose and maltose but not lactose or saccharose and only grows at blood
temperature, thus distinguishing it from the M. catarrhalis which will not ferment
any of these sugars. It is scarcely pathogenic for laboratory animals, with the
exception of the mouse and guinea-pig, when intraperitoneal injections but not
subcutaneous ones give results. Intradural injections give results. The cultures
die out very rapidly, so that it is necessary to make transfers every one or two
days. The Meningococcus has been isolated from the nasal secretions of patients.
The possibility of these organisms being the M. catarrhalis must be considered.
Of the greatest importance is the examination of the naso-pharyngeal material
of those who have been in contact with a patient. These healthy carriers are im-
portant. To examine such people introduce a bent wire applicator with sterile
cotton tip past the soft palate so as to get material from the naso-pharynx. The
material should be immediately inoculated on blood or serum agar and quickly put
in the 37°C. incubator.

Flexner has shown that in monkeys, which are susceptible to the disease, in-^
jections of cultures of M. intracellularis into the spinal canal is followed by migra-
tion of the cocci to the nasal cavity both free and in phagocytic leukocytes.

The Meningococcus has a very slight resistance to sun or drying so
that its aerial transmission seems doubtful. It is supposed to effect an
entrance by the nares, thence reaching the cerebral meninges. Infec-
tion is probably by direct contagion. Several cases have been reported
where with a high leukocytosis the cocci have been found in the poly-
morphonuclears of blood smears and in cultures from the blood. (In
about 25% of blood cultures where from 5 to 10 c.c. are employed.)

By the use of initial injections into horses of killed cultures followed by alternate
injections into horses of living diplococci, then seven days later of an autolysate
made from different strains; seven days later again injecting living diplococci; thus
alternating material every week, an antiserum of value has been obtained by Flexner.
The immunization requires about one year. In using, withdraw about 20 c.c. of
patient's cerebrospinal fluid with a syringe, and then inject, through the same needle,
an equal quantity of the serum. The injection is repeated every day for three or
four days.

As the result of extensive study of the relation of the Meningococcus to cerebro-
spinal fever in connection with epidemics in the European war zone many authorities
consider the causal relation of the organism to the disease as unproven. It is stated
that intraperitoneal injection of huge numbers of meningococci, cultivated in the
cerebrospinal fluid, failed to cause symptoms in monkeys while intraperitoneal
injection of a filtrate of fluid just removed from a patient caused symptoms in the
monkey similar to cerebrospinal fever.

The history of hog cholera is cited by Hort and others to show that our long-stand-
ing views as to the~relstibnship of the hog cholera organism (B. aertryk] had to be
abandoned in favor of a filterable virus etiology, The possibility of a filterable
virus being the cause of cerebrospinal fever is suggested but the point is emphasized

70 STUDY AND IDENTIFICATION OF BACTERIA

that the presence of meningococci in a person has a value as indicating the presence
of the virus, so that such examinations should continue, but they insist upon the
futility of means for eradicating meningococci from the nasopharynx by disin-
fecting'solutions.

For diagnosis, make smears and cultures from cerebrospinal fluid.
The sediment from the centrifuged material gives better results. In
tuberculosis the lymphocytes preponderate; in cerebrospinal meningitis
the polymorphonuclears.

It has been stated that a point of difference between the phagocytosis with the
gonococci and the meningococci is that the meningococci invade and at times destroy
the nucleus of the polymorphonuclear, which is not true of gonococci. The appear-
ance of large phagocytic endothelial cells, often containing polymorphonuclears, in

FIG. 1 6. — Diplococctis intracdlularis meningitidis and pus cells. (Xiooo.)

(Williams.}

the centrifuged cerebrospinal fluid is a favorable prognostic sign. At times there
does not appear to be any relation between the number of phagocytic polymorpho-
nuclears and the severity of the case.

Vincent has recommended a precipitin test for epidemic cerebrospinal meningitis
which has the advantages of being simple and more immediate than cultures and of
particular value in those cases when meningococci cannot be found in the smears or
in cultures from the cerebrospinal fluid. It is performed by adding i or 2 drops
of antimeningococcic serum to a tube of fresh cerebrospinal fluid which has been
clearecf by centrifugalization for ten to fifteen minutes. After adding the serum the
tube is placed in the incubator at S2°C. for two to five hours together with a control
tube. The formation of a precipitate (turbidity) shows a positive test.

Micrococcus Catarrhalis (Seifert, 1890). — This organism has been
specially studied by Lord. It resembles the Meningococcus strikingly
and can only be differentiated by cultural procedures. It grows on

MALTA FEVER 71

plain agar and at room temperature, and does not produce acid in glu-
cose media. It not only occurs in the nasal secretions of healthy people,
but appears to be responsible for certain coryzas and bronchial affec-
tions, resembling influenza. It also is responsible for certain epidemics
of conjunctivitis.

The original cultures may show only slight growth whereas the subcultures
prove luxuriant.

The colonies are larger, more opaque, and have a more irregular wavy border
than the round colonies of the Mening&coccus.

The colony tends to be easily picked up from the plate with the loop. M. catar-7~s;
rhalis grows well at 22° C. after several days, while the Meningococcus requires body^"""
temperature. It does not ferment with acid production any of the sugars.

Micrococcus Melitensis (Bruce, 1887). — This is the organism of
Malta or Mediterranean fever, sometimes called undulant fever, on
account of successive waves of pyrexia running over several months. UJ
The disease has a very slight mortality (2%), and the lesions are chiefly
of the spleen, which is large and diffluent. The organisms can best be (/j
isolated from the spleen or blood.

M. melitensis is only about 0.3^1 in diameter. The characteristics are its very
small size and the dew-drop minute colonies on agar, which at incubator temperature
only show themselves about the third to the sixth day. It is nonmotile and Gram-
negative. In bouillon there is a slight turbidity. Gelatine growth is very slow and
there is no liquefaction. Litmus milk becomes more blue after a week so that there
is an alkaline action. Indol is not produced. The optimum reaction of media is
+ 0.8. It grows best at 38°C.

Many laboratory infections have been recorded.

The organism occurs in peripheral circulation, it having been cultivated from
blood very successfully by Eyre. He takes blood at the height of the fever, and in
the afternoon. Formerly it was customary to isolate by splenic puncture.

Infection is chiefly by means of the milk of infected goats. The organisms are
excreted in the urine of patients, and a diagnostic point is to make plates from the
urine. Such urine applied to abraded surfaces causes infection.

The serum of patients shows agglutinating power as early as the fifth day of
the disease, and this may persist for years after recovery. Nicolle has advised using
serum heated to 56°C. for thirty minutes for the agglutination test, nonspecific agglu-
tinins being thereby destroyed. Carriers may be of importance in Malta fever and
are best detected by agglutination tests.

A high mononuclear increase may be found in this disease.

Horses, cows, asses, as well as goats, are susceptible. It is very difficult to
infect rabbits, mice and guinea-pigs. Monkeys have been chiefly utilized in ex-
perimental work.

It would appear as if there were other organisms closely related to M. melitensis
and a great deal is now being written as to confusing serum reaction from the use
of M. paramelitensist

4

72 STUDY AND IDENTIFICATION OF BACTERIA

What may be deemed proof positive of goats' milk transmission is
the practical disappearance of the disease among the naval and mili-
tary forces of Malta, as the result of boiling the milk, while still con-
tinuing among native civilians not boiling their milk. Bassett-Smith
has noted that in 1905 there were 798 cases among civilians and 245
naval cases. In 1907 there were 457 cases among civilians and only
twelve cases in the naval forces.

There are however occasional cases which Shaw has considered as due to carriers.
As the organisms are excreted in faeces as well as in urine, and as the course of the
disease is so protracted, as well as the convalescence, it would seem that the carrier
factor should be of more importance than facts would justify.

Mohler has noted in Texas, where the disease has existed for
twenty-five years, that the Mexican goatherds boiled their milk and
hence were rarely infected.

The souring of milk does not destroy the germs of the disease, hence transmission
may be brought about by butter and cheese.

Malta fever was stamped out of Port Said by destroying'all infected goats.

Infection may occur, i. by the stomach atrium (usual), 2. contam-
inated dust reaching lungs, 3. by subcutaneous infection.

CHAPTER VI

STUDY AND IDENTIFICATION OF BACTERIA. SPORE-
BEARING BACILLI. KEY AND NOTES

A. Grow aerobically.

1. Stab culture in gelatin has branches growing out at right angles to line of stab,
(a) Has no membrane on bouillon or liquefied gelatin. Projecting branches

from line of stab only at upper part of line of growth. Absolutely non-
motile. Ends shaiplyjnit across or concave. ANTHRAX GROUP.
(6) Has thick whitisfTniembrane on bouillon and surface of liquefied gelatin.
Projecting branches all along the line of stab. Sluggishly motile. MY-
COIDES GROUP. (B. mycoides. B. ramosus.)""

2. Stab cultures in gelatin do not show projecting branches.

(a) Potato cultures do not become wrinkled. At first slightly moist, later
dry and mealy. SUBTILIS GROUP. (Hay bacillus group.) Actively
motile with more or less square ends and a central spore which is of the
same diameter or only slightly larger than the bacillus. The yellow sub-
tilis is at times found in water. The colonies on potato are of a cheese-
yellow color. The bacilli are very large and show a sluggish, worm-like
motion.

B. megatherium often shows a granular or beaded appearance in a Gram
preparation. The narrow spores are never central, usually between center
and end, and rather elongated. It most nearly resembles the sporulating
bacillus of malignant oedema but if the spore is quite terminal and bulging
may resemble B. tetani.
Cultures of B. megatherium are somewhat similar to B. coli colonies.

(&) Potato cultures at first even growth but after a few days become wrinkled.
VULGATUS GROUP. (Potato bacillus.)

B. vulgatus shows marked wrinkling, like intestinal coils. B. mesentericus
show slight wrinkling and a network-like appearance.
Two water bacilli belonging to this group are the B. mesentericus fuscus
(brown growth) and B. mesentericus ruber (red growth).

NOTE. — The following cultural characteristics are common to all the above spore
bearers.

1. Liquefaction of gelatin.

2. Milk slowly and incompletely coagulated with very little change in reaction.
Later the coagulum is digested.

3. No gas in either glucose or lactose.

4. No indol.

5. All are Gram-positive.

6. All digest blood-serum. ^^

W 73

74

STUDY AND IDENTIFICATION OF BACTERIA

NONPATHOGENIC SPORE-BEARING AEROBES ON AGAR
Modification of table of Gruner and Fraser.

Gray white

1 Chalk like

( Edges feathery

Surface gummy f Yellow

\ White

Yellow

Surface moist

Dirty gray

and exuberant

White and

wrinkled.

B. subtilis (motile).

B. ellenbachensis (non-motile).

B. cretaceus.

B. mycoides.

B. ochraceus.

B. mesentericus.

B. ruminatus.

B. graveolens,.

B. vulgatus.

NOTES. — B. subtilis has square ends and central spores, not causing bulging.

B. megatherium has spore toward one pole which may be terminal.

B. vulgatus is long and slender. Slightly oval spores.

B. mesentericus varies. Usually short with rounded ends and central bulging
spores.

B. ellenbachensis has rounded ends, oval spores and shows granule formation
(beaded) — resembling diphtheroids.

All of these grow well at room temperature, optimum 3o°C.
B. Grow only anaerobically.

1. Rods very little swollen by centrally situated spores,
(a) Motile. B. cedematis maligni. (Gram-negative.)
(6) Nonmotile. B. aerogenes capsulatus. (Capsule.)

2. Spores tend to be situated between center and end.
(a) No liquefaction of gelatin. B. butyricus.

(6) Gelatin liquefied slowly.

B. botulinus. Milk not coagulated.

B. anthracis symptomatici.

B. enteritidis sporogenes. Milk coagulated with abundant gas.
(c) Gelatin liquefied rapidly. B. cadaveris sporogenes. Very motile.

3. Spores situated at end of rod. Drum-stick sporulation. TETANUS GROUP.

The following table taken from Lehmann and Neumann, based on
pathogenic effects, is of great practical value. After inoculation of
some animal subcutaneously with the suspected material we have:

A. No particular symptoms at site of inoculation.

Absorption of the soluble toxin causing:

(1) General symptoms of tetanus. B. tetani.

(2) Botulism poisoning symptoms. Pupillary symptoms. Paralysis of
tongue and pharynx. Cardiac and respiratory failure.

B. Local symptoms marked at site of inoculation. Hemorrhagic emphysema tous
oedema.

(i) Motile.

(a) Gram-negative. B. cedematis maligni.

(6) Gram-positive. ^anthracis symptomatici.

ANTHRAX

75

(2) Nonmotile.

B. aerogenes capsulatus, B. phlegmonis emphysematosse (Frankel) or
B. perfringens.

SPORE-BEARING AEROBES

Bacillus anthracis (Pollender discovered 1849. Davaine recognized
nature 1863. Koch proved 1876).— Of the aerobic_spore-bearing
bacilli this is the only one of particular medical importance""

Anthrax is an important disease in domesticated animals, especially sheep andcattle.
The characteristic postmortem change in animals is the greatly enlarged, friable,
mushy gpleen. Man is much less susceptible than these animals, but is more so
than the goat, horse, or pig. The 41gerian^sheep has a high degree of immunity,

FIG. 17. — Anthrax bacilli. Cover-glass has been pressed on a colony and then fixed
and stained. (Kolle and Wassermann.)

as has the white rat. The brown rat is quite susceptible as are also guinea-pigs, mice
and rabbits. The disease in man chiefly occurs among those working with hides,
wool, or meat of infected cattle. The two chief types in man are: i. Malignant
pustule and 2. Woolsorter's disease. An intestinal type is also recognized.

Malignant pustule results from the inoculation of an abrasion or cut;
thus it frequently shows on the arms and the backs of those unloading
hides. It first appears as a pimple, the center of which becomes vesicu-
lar, then necrotic.

A ring of vesicles surrounds this central eschar and a zone of congestion, the
vesicles. The lymphatics soon become inflamed as well as neighboring glands.
If the pustule is not excised andtleath occurs, there is not much enlargement of the

STUDY AND IDENTIFICATION OF BACTERIA

spleen and the bacteria are not abundant in the kidneys, etc., as with animals.
Man seems, to die from toxaemia rather than a septicaemia.

In woolsorter's disease there is great swelling and oedema of the bronchial
and mediastinal glands. The lungs show oedema, which about the bronchi is
hemorrhagic.

The bacillus is 5 to 8p by i to ij^/i and nonmotile. In cultures it
has square cut or concave ends and is often found in chains, but in the
blood of an infected animal the free ends of the rods are somewhat
rounded. It is Gram-positive. Colonies, by interlacing waves of
strings of bacteria, show Medusa head appearance. For cultural char-
acteristics see key. Spores develop best at a temperature of 3o°C. and

FIG. 1 8. — Anthrax bacilli growing in a chain and exhibiting spores. (Kolle and

Wassermann.)

do not form at temperatures above 43°C.
placed. They stain with difficulty.

They are oval and centrally

•

Stiles thinks that animals are infected by eating the bones of animals which have
died of anthrax, cutting buccal mucous membrane, and so becoming infected.
Spores do not form in an intact animal body, but they do form ^ after ji postmortem
or the disintegration of the body by maggots. For this reason it is better not to
open up the body of the animal, but to make the diagnosis by cutting off an ear.
Dried spores will live for years and will withstand boiling temperature for hours.

In vaccinating animals against anthrax, Pasteur used two vaccines. The first
is attenuated fifteen days at 42.5°C. The second, attenuated for only ten days, is
given twelve days later.

Various bacteria, especially B. pyocyaneus, show marked antagonism to B.
^anthracis. Pyocyanase digests the anthrax bacillus and has been used to cure

iN ot-iirviolc info^i-orl TirifV* *» TifV» TO v

animals infected with anthrax.

SYMPTOMATIC ANTHRAX

77

In taking material from a malignant pustule before excision, be care*
ful not to manipulate it roughly, as bacteria may enter the circulation.
Make cover-glass preparations, staining by Gram. Make culture on
agar. Blood cultures are usually only positive later in the disease.
Inoculate a guinea-pig or a mouse subcutaneously.

The guinea-pig dies in about forty-eight hours and shows an oedematous gelatinous
exudate at site of inoculation. The blood is black and swarms with anthrax bacilli.
It is the best example of a septicaemia.

Anorganism with a central spore and morphologically resembling B. anthracis,
but motile, has been reported as occurring in the stools of pellagrins. Gelatin stabs
show a cup-shaped liquefaction in about five days. No change in milk. The

FIG. 19. — Bacillus anthracis in blood of rabbit. (Coplin.)

colonies are slimy and opaque. The organism is said to be agglutinated by the
serum of pellagra cases. The name B. maydis has been given to it.

There is an anaerobic spore bearer, called the bacillus of symptomatic
anthrax, blackleg or quarter-evil, which causes a rapidly developing
emphysematous swelling, with a dark color of the thighs. It is called
B. chauwei. It has bulging, slightly oval spores at one end, but they
are not distinctly terminal as with tetanus spores. It affects sheep
and cattle but not man. It is a soil organism like those of tetanus,
malignant oedema and gas gangrene.

There is also an aerobic spore-bearing bacillus called B. anthracoides,
which differs morphologically from anthrax solely in its rpunded_erids
jn Culture. Its growth is more rapid and it liquefies gelatin' more
energetically.

78 STUDY AND IDENTIFICATION OF BACTERIA

SPORE-BEARING ANAEROBES

There are four very important pathogens in this group — that of gas
gangrene; that of malignant oedema; that of botulism, and the organism
of tetanus.

The B. enteritidis sporogenes is of importance in connection with indications of
faecal contamination of water. In connection with B. aero genes capsulatus, there is
some question as to whether the extensive oedema produced by it may not usually

be from a terminal or cadaveric infec-
tion. At any rate necrotic material
seems necessary.

It should be stated that our knowl-
edge of the differential cultural charac-
teristics of anaerobes is unsatisfactory.
The exact methods which are in use
for aerobes have not been applied to
anaerobic organisms.

To Cultivate Anaerobes. — Prob-
ably the apparatus giving the most
perfect anaerobic conditions is the
Novy jar, in which the air has been
replaced by hydrogen. The diffi-
culties attending the method are:

i. Unless a special apparatus (Kipp's)
is at hand, there may be difficulty in pre-
FIG. 20. — Novy jar. venting the sulphuric acid from frothing

over when poured on the zinc. It should,
at first, be added in small quantities at a time — well diluted (i to 6).

2. Various wash-bottles are required: one containing silver nitrate solution for
traces of AsH3 and one with lead acetate for H2S and another with pyrogallic acid
and caustic soda for any oxygen that may come over.

3. Mixtures of hydrogen and air explode. Consequently, in determining whether
all air has been expelled and in its place an atmosphere of hydrogen exists, it is
necessary to see if the escaping gas burns with a blue flame. Unless this is collected
in a test-tube and examined, we may have an explosion.

4. Except in a large laboratory, where the apparatus is set up and ready for use,
too much time would be required.

5. Simpler methods appear to give as good results.

In Tarozzi's method, pieces of fresh sterile organs are added to
bouillon. Pieces of kidney, liver, or spleen are best suited. After
adding the tissue the media may be heated to 8o°C. for a few minutes

CULTTJRING ANAEROBES

79

without interfering with the anaerobic condition producing properties
of the fresh tissues. This method is practically the same as that recom-
mended by Smith (see Tetanus). This is also a feature of Noguchi's
method of culturing Treponema pallidum.

The Method of Liborius

In this it is necessary to have a test-tube containing about 4 inches of a i%
glucose agar. Glucose acts as a reducing agent and furnishes energy. It is con-
venient to add about i/io of i% of sulphindigotate of soda; the loss of the blue color
at the site of the colony enabling us to pick them
out. The tube of agar should be boiled just be-
fore using' to expel remaining oxygen from the
tube. Now rapidly bring down the temperature
to about 42°C., by placing the tube in cold water,
and inoculate the material to be examined. A
second or third tube may be inoculated from the
first, just as in ordinary diluting methods for plate
cultures. Having inoculated the tubes, solidify
them as quickly as possible, using tap water or
ice-water. The anaerobic growth develops in the
depths of the medium. Some pour a little sterile
vaseline or paraffin or additional agar on the top
of the medium in the tube as a seal from the air.
Others have recommended the inoculation of some
aerobe, as B. prodigiosus, on the surface. This
latter method is not advisable. A deep stab
culture is often sufficient.

The same technic can be applied to gelatin
cultures for anaerobes, pouring in at the comple-
tion of the inoculation an inch or so of melted
glucose agar to act as a stopper for the gelatin
layer below.

FIG. 21. — Arrangement of
tubes for cultivation of anae-
robes by Buchner's method.
(Mac NeaL)

The Method of Buchner

In this method i gram each of pyrogallic acid
and caustic potash or soda for every 100 c.c. of
space in the vessel containing the culture is used
to absorb the oxygen. It is convenient to drop
in the pyrogallic acid; then put in place the in-
oculated tubes or plates; then quickly pouring in the amount of caustic soda, in a
10% aqueous solution, to immediately close the containing vessel. A large test-
tube in which a smaller one containing the inoculated medium is placed, and which
may be closed by a rubber stopper, is very convenient. A good rubber-band fruit
jar is satisfactory. A desiccator may be used for plates. An excellent method for

80 STUDY AND IDENTIFICATION OF BACTERIA

anaerobic plates, either in a desiccator with the pyrogallic acid and caustic soda, or
less satisfactorily in the open air, is to sterilize the parts of the Petri dish inverted;
that is, the smaller part is put bottom downward in the inverted cover (as one would
set one tumbler in another). Then, in using, unwrap the Petri dish, lift up the inner
part, pour in the inoculated medium into the upturned cover. Then immediately
press down the inner dish, spreading out a thin film of the medium between the two
bottoms.

Zinsser has originated a very satisfactory method for plate cultures of anaerobes,
which is shown in Fig. 8.

Secure two small crystalh'zing dishes, about 3 and 4 inches in diameter by i inch
depth and sterilize as for Petri dishes. Pour the inoculated agar into the smaller
of the dishes or one can smear the surface of poured glucose agar with the material
to be plated out. In the bottom of the larger dish place the dry pyrogallic acid,
then invert the smaller dish with the agar surface over it. Quickly pour a 5%
solution of caustic soda into the space separating the sides of the inverted smaller
dish and the upright larger dish, to a depth of % inch and, as it is dissolving the
pyrogallic acid, very speedily superimpose paraffin oil on the soda solution to make
an air-tight seal.

J. H. Wright's Method

Make a deep stab culture in glucose agar or gelatin, preferably boiling the media
before inoculating. Then flame the cotton plug and press it down into the tube so
that the top lies about three-fourths of an inch below the mouth of the test-tube.
Next fill in about one-fourth of an inch with pyrogallic acid; then add 2 or 3 c.c. of
a 10% solution of caustic soda, and quickly. insert a rubber stopper. This method
is one of the most convenient and practical, and is to be strongly recommended.

Method of Vignal

In this a section of glass tubing (1/4 in.) is drawn out at either end, as in making a
bacteriological pipette, with a mouth-piece containing a cotton plug. The liquid
agar or gelatin is then inoculated and the medium drawn up into the tube by suction
with mouth or better with a rubber bulb. In a very small flame the capillary nar-
rowings are sealed off, and we have inside the tube very satisfactory anaerobic con-
ditions. To get at the colonies, file a place on the tube and break at this point.

To obtain material for examination and isolation in pure culture from the deep
agar stab-tube, it is best to loosen the medium at the sides of the tube with a heated
platinum spud or a flattened copper wire. Then shake the mass out into a sterile
Petri dish. It is dangerous to break the tubes with a hammer as some do.

With those anaerobes which produce gas in glucose agar the split in the column
of medium enables one to introduce a fine sterile capillary pipette to the site of a
colony and by releasing pressure on the rubber bulb to draw up into the tip of the
tube material for investigation.

A Combination Method

Recently as shown in the illustration in Fig. 7, I have been combining various
methods so that very satisfactory anaerobic conditions are obtained. First, a

GAS GANGRENE 8 1

deep agar stab of freshly sterilized glucose agar is made and inoculated. The sur-
face of this is then covered with sterile paraffin oil. The proper amount of pyro-
gallic acid is then deposited in a salt mouth bottle. The rubber stopper with the
glass and rubber tubing is then firmly pushed in and connection made with a filter
pump.

In five to ten minutes almost all the air will be exhausted when the Hofmann
clamp is screwed up tight and the bottle disconnected from the vacuum pump.
The glass tubing end is then inserted into a graduate holding 10% caustic soda
solution, the Hofmann clamp unscrewed, and the necessary amount of caustic soda
having been run in, as noted under Buchner method, we again close the screw clamp
and incubate.

GENERAL CONSIDERATIONS OF PATHOGENIC ANAEROBES

Dean and others, working with gas gangrene wounds, have brought
out some very practical points in connection with the three important
anaerobes found in such wounds, viz.: B. aero genes capsulalus, B. adem-
atis maligni and B. tetani.

They found an egg broth, made by shaking up the white and yolk of i egg in
300 c.c. water a most excellent culture medium. This medium was tubed and
sterilized, after which it was liberally inoculated with material from the wounds.
After inoculation the tube was heated to 8o°C. for one-half hour and then incubated
anaerobically.. The gas bacillus was present in abundance in such cultures after two
or three days' incubation, while the bacillus of malignant oedema later on became the
predominant organism. The tetanus bacillus only appeared after a prolonged period
— seven to ten days. The bacillus of malignant oedema grew best on Dorsett's egg
medium and in two days began to liquefy the slant with a bluish-black discolora-
tion. The growth at first was profuse and creamy white. On glucose agar there
was much less gas production than with the gas bacillus. The malignant oedema
organisms were as a rule Gram-negative on glucose agar but they were distinctly
Gram-positive on Dorsett's medium. The spores were oval in shape, usually
located near the end of the rod. The gas bacillus grew well on Dorsett's medium
but less vigorously. In glucose agar stabs so much gas was formed that the
cotton plug and much of the culture medium tended to be expelled from the
tube. The spores form well on Dorsett's medium but not in glucose agar and
show as oval bodies distending the central portion of the rod.

Subcutaneous inoculation of the gas bacillus and that of malignant
oedema rarely produced death in guinea-pigs.

As regarded the tetanus bacillus they tried various methods of bacteriological
diagnosis. The examination of smears from wounds was unsatisfactory in search
for "drum-stick" spores. In broth cultures the spores were not present until after
several days and in mixed cultures it was difficult to be sure that the terminal spores
were those of tetanus and not atypical malignant oedema spores. The best method
6

82 STUDY AND IDENTIFICATION OF BACTERIA

was to inject guinea-pigs in the subcutaneous tissues of the left chest with i or 2 c.c.
of mixed broth culture. In two or three days stiffness of the left forelimb was observed
soon becoming quite stiff and extended. The spasm extends to other limbs and
death occurs in one or two days. There is no evidence of marked inflammation at
the site of inoculation.

There have been a number of cases of delayed tetanus often asso-
ciated with operations done a month or two after the original wound
infection so that it is recommended to give antitetanic serum before
operation on such cases.

B. (Edematis Maligni (Pasteur, 1877). — This is the vibrion septique
of Pasteur. It is found in garden soil and in street sweepings. It is
the cause of an acute cellular necrosis attended with serous sanguino-
lent exudation and with more or less emphysema. The organism only
becomes generalized in the blood about the time of death and post-
mortem. Therefore, it is not a septicaemia, as is anthrax. The bacil-
lus is an organism about the size of anthrax (*jn by 0.8), but is narrower
and does not have the same square cut or dimpled ends. Furthermore,
it is motile, Gram-negative and an anaerobe. The guinea-pig is very
susceptible, and about the time of death and postmortem there may be
seen long flexile motile filaments, 15 to 40^1 long, which move among
the blood-cells as a serpent in the grass (Pasteur).

In cultures it grows out very slightly from the line of stab, giving a jagged granular
line, differing from tetanus. Spores form best at 37°C. — requiring about forty-
eight hours. It liquefies gelatin. In examining an exudate from a suspected case-
note the presence of spores centrally situated. Inoculate a guinea-pig. Death
occurs in about two days. There is intense hemorrhagic emphysematous oedema at
the site of inoculation, the cedematous fluid however does not show spores. The
bacilli do not appear in the blood until about the time of death and it is an assistance
in diagnosis to put the dead body of the guinea-pig in the incubator for a few hours.
The subcutaneous tissue contains fluid and gas. There is present the foul odor of
an anaerobe. Examine for the long filaments showing flowing motility. Be sure
to stain by Gram. (Negative.) For cultures, heat the material (either from a
wound or from a guinea-pig) which shows spores to a temperature of 80° C. for
from fifteen minutes to one hour. Then inoculate glucose agar stab culture and grow
anaerobically. Courmont differentiates anthrax from malignant oedema by in-
jecting into ear-vein of rabbit. The injection of malignant oedema in this way,
instead of subcutaneously, tends to immunize.

B. Botulinus (Van Ermengem, 1896). — This is the organism which
produces botulism, a form of meat poisoning. It is a spore-bearing
anaerobe and must not be confused with another organism, a non-
sporing aerobic bacillus, associated with meat poisoning — the B.

BOTULISM

enteritidis of Gartner. The spores are at the end and are not very
resistant; a temperature of 8o°C. often killing them.

FIG. 22. — Bacillus of botulism. (Kolle and Wassermann.)

In botulism the meat becomes infected after the animal has been slaughtered;
in Gartner meat poisoning the cow meat was infected at the time of slaughter — it
was from a sick animal. Thorough cooking of the meat protects against botulism
but not certainly against Gartner meat poisoning.

FIG. 23. — Symptomatic anthrax (Rauschbrand) bacilli showing spores. (Kolle

and W assermann.)

There are dysphagia, paralysis of eye-muscles, and cardiac and respiratory
symptoms (medulla). The symptoms are due to the elaboration of a soluble toxin
of the same nature as that of diphtheria and tetanus. There is no fever and con-
sciousness is preserved.

84 STUDY AND IDENTIFICATION OF BACTERIA

An antitoxin which it is stated has therapeutic value in botulism has been pre-
pared in the usual way by Kempner. Without serum treatment death occurs in
about 40% of cases and takes place between twenty-four and forty-eight hours.

The bacillus has been isolated from sausage and ham. It is a rare cause of food
poisoning, most of such cases being the result of paratyphoid or enteritidis infections.
It is a large bacillus — 5 to ic/xXiM- It is slightly motile and stains by Gram.
It produces gas in glucose media. It grows best at 22° and only slightly at 37° —
hence it is dangerous only from its soluble toxin, the bacilli not developing to any
extent in the body.

For this reason botulism patients are not a source of danger, it is the infected
meat alone which causes the disease. On the contrary where the meat poisoning
is due to the Gartner or paratyphoid group infection may take place from the
patient's discharges. Botulism is an intoxication, not an infection.

When the toxin is introduced, it requires a period of incubation of
twelve to twenty hours. Symptoms of gastrointestinal disorder may
come on shortly after the ingestion of the toxin containing food, these
however are not the specific manifestations, as are the eye symptoms,
etc.

An important point is that ham may not appear decomposed and yet contain
many bacilli and much toxin. It is a very potent toxin — as little as Ho 00
c.c. may kill a guinea-pig. In man the toxin is apparently absorbed from the
alimentary canal, thus differing from most toxins as well as venoms which are
usually harmless when introduced by mouth.

For diagnosis inject an infusion of the ham or sausage which was eaten of into a
guinea-pig, and characteristic pupillary symptoms with death by cardiac and
respiratory failure will result.

Cultures may be made in glucose agar.

The culture is disrupted by gas. Incubation at room temperature and in the
dark is necessary. There is a rancid odor. The characteristic point is the pro-
duction of a powerful soluble toxin which produces symptoms when no bacilli are
present.

B. Tetani (Nicolaier, 1885; Kitasato, 1889).— This is the most im-
portant'organism of the anaerobic spore bearers. Its characteristics
are the tetanic symptoms produced by the toxin and the strictly termi-
nal drum-stick spores.

Spores are difficult to find in material from wounds infected with tetanus, but
readily develop in cultures. See notes under general consideration of pathogenic
anaerobes. Prior to the formation of spores the organism is a long thin bacillus
(4Xo.4/u). It is motile and Gram-positive. It liquefies gelatin slowly and does
not coagulate milk. The stab culture in glucose agar shows pine-tree growth.
Colonies on agar plates show as fleecy clouds and microscopically as felted filaments.

Theobald Smith recommends growing it in fermentation tubes containing ordinary
bouillon, but to which a piece of the liver or spleen of a rabbit or guinea-pig has been

TETANUS 85

introduced at the junction of the closed arm and the open bulb. By this method
spores develop rapidly in from twenty-four to thirty-six hours. Sporulation is
most rapid at 37°C. As there is always liability to postmortem invasion of viscera
by ordinary saprophytes, Smith recommends that great care be taken not to handle
the animal roughly in chloroforming and in pinching off pieces of the organ at
autopsy. The animal must be healthy, and the tubes to which the piece of tissue
is added must be proven sterile by incubation. Smith calls attention to the un-
certainty of the temperature at which tetanus spores are killed. He shows that
some require temperature only possible with an autoclave. In view of the danger
of tetanus, it is advisable to carefully autoclave all material going into bacterial
vaccines, such as salt solution, bottles for holding, etc.

Tetanus seems to grow better in symbiosis with aerobes; hence a
lacerated dirty wound with its probable contamination with various
cocci, etc., and its difficulty of sterilization, offers a favorable soil.
The tetanus bacillus gives rise to one of the most powerful poisons
known; it is a soluble toxin like diphtheria toxin, and it is estimated
that J^joo grain is fatal for man.

There are 2 toxins, tetanospasmin and tetanolysin, but the former
is the important one. Tetanus toxin is twenty times as poisonous as
dried cobra venom.

The antitoxin is produced by injecting horses with increasing doses of tetanus
toxin, following a preliminary dose of 5000 antitoxin units. An important point
is that a horse used for the production of diphtheria antitoxin may become infected
with tetanus and his blood contain enough tetanus toxin to kill. A number of
children in St. Louis died of tetanus as the result of such an accident.

Rosenau has established an antitoxin unit for tetanus which has the power of
neutralizing 1000 minimal lethal doses. Practically, it is ten times the least
quantity of antitetanic serum necessary to protect the life of a 350 grams
guinea-pig from a test dose of tetanus toxin furnished by the hygienic laboratory.
(The necessity of some definite unit is apparent when tests have shown that serum
stated to contain 6,000,000 units per c.c. only had a value of 90 of the official
American units.) Consequently it is a unit ten times as neutralizing as the diph-
theria antitoxin one. The antitoxin of tetanus is less efficient than that of diph-
theria for the following reasons:

1. There is about three times as great affinity in vitro between diphtheria toxin
and antitoxin as is the case with tetanus.

2. The tetanus toxin has greater affinity for nerve cells than for antitoxin.

3. Treatment with antitoxin is successful after symptoms of diphtheria appear.
With tetanus it is almost hopeless after the disease shows itself. Hence the impor-
tance of the early bacteriological examination of material from a suspicious wound
(rusty nail).

4. The tetanus toxin ascends by way of the axis cylinder, and the antitoxin
being in the circulating fluids cannot reach it, wheras with diphtheria both toxin
and antitoxin are in the circulation. Diphtheria also selects the cells of paren-

86 STUDY AND IDENTIFICATION OF BACTERIA

chymatous and lymphatic organs which are more tolerant of injury than the nerve
cells. The dose of tetanus antitoxin as a prophylactic is 1500 units; as a curative
agent 5000 to 20,000 units. Recent experience shows that it should be injected
intravenously when symptoms have manifested themselves.

That the disease is due to toxin is shown not only experimentally, but also if
spores are carefully freed of all toxin by washing, and then introduced they do not
cause tetanus — the polymorphonuclears engulfing them. The importance of the
presence of ordinary pus cocci in a tetanus wound may be that the activity of the
leukocytes in phagocytizing them allows the tetanus bacillus to escape phagocytosis.
This would also explain the importance of necrotic tissue in a lacerated wound —
the phagocytes taking this up instead of tetanus bacilli. The toxin is digested by
the alimentary canal juices and infection by that atrium is improbable. The in-
fection occurs especially through skin wounds, and also from those of mucous mem-
brane. While tetanus is like diphtheria, a disease in which the bacilli are localized

FIG. 24. — Tetanus bacilli showing end spores. (Kolle and Wassermann.)

and do not spread, yet recently Richardson has obtained tetanus bacilli in pure
culture from the tributary lymphatic glands of a "rusty nail" wound of foot. The
cultures inoculated into root of tail of a white rate caused the rat's death in forty-
eight hours with typical "seal gait" attitude of tetanus in rats.

The usual period before symptoms occur is fifteen days. The shorter the period
of incubation, the more probably fatal the disease. The horse is the most susceptible
animal, next the guinea-pig, then the mouse. Fowls are practically immune.

In examining for tetanus, scrape out the granulation tissue or foreign
material from the suspected wound with a sterile Volkmann spoon and
insert in a pocket made with scissors in the subcutaneous tissues of the
thigh of a guinea-pig. Animal inoculation is the practical method.
One may also put some of the suspected material in a Loffler's serum
tube. Place in the incubator, under which conditions the cocci and
other aerobes grow luxuriantly and enable the tetanus bacillus to de-

THE GAS BACILLUS 87

velop. From day to day smell the culture, and if an odor similar to the
penetrating, sour, foul smell of the stools of a man who has been on a
debauch be detected, it is suspicious. The nondevelopment of a foul
odor is against tetanus. Also make smears from the material and ex-
amine for drum-stick spores. If these are found, heat the material
to 8o°C. for one-half hour, to kill nonsporing aerobes and faculative
anaerobes, and then inoculate a deep glucose agar tube and cultivate
by Wright's method. The fusiform lateral outgrowth about the middle
of the stab is characteristic.

A more rapid method is to draw up the material, provided it be pus (tissue scrap-
ings may be emulsified in sterile salt solution) into a capillary bulb pipette. Then
seal off the end and heat the capillary bulb pipette and its contents in a water-bath
at 8o°C. for fifteen minutes. Next break off the sealed tip and stick the pipette into a
deep tube of glucose agar. When the point reaches the bottom, force out the material
along the line of the stab as the pipette is withdrawn. Cover the surface of the
agar with sterile liquid petrolatum and incubate. Better anaerobic conditions
obtain where the Buchner or Wright method is employed.

Tetanus produces no gas. Material for examination is best obtained with a bulb
pipette (containing a little sterile salt solution) which is plunged into the agar and the
salt solution forced out and drawn in where a proper growth is noted.

Spores form in thirty-six to forty-eight hours. In injecting test animals it is
advisable to divide the material to be injected into two portions; one animal is
injected with the material alone, the second animal with tetanus antitoxin at the
same time the material is injected. Only the first animal dies with tetanic symptoms.

B. Aerogenes Capsulatus (Welch, 1891). — This bacillus is apparently
widely distributed. It is possibly the same organism as Klein's B.
enteritidis sporogenes, which is constantly present in faeces. It is a
large capsulated organism, which does not form chains. Spores are
produced on blood-serum. These are frequently absent on other
media. It is questioned whether its pathogenicity is other than ex-
ceedingly feeble, the presence of the bacillus in emphysematous find-
ings at postmortem being attributed to terminal or cadaveric invasion.

Cases, however, in the Philippines, have been reported following carabao horn
wounds, in which most serious and fatal results attended emphysematous lesions
showing this bacillus. The isolation of a Gram-positive bacillus from a lacerated
wound discharge, even in the absence of emphysema, is almost diagnostic.

In milk cultures we have coagulation and from the subsequent development
of gas the disruption of the coagulum into shreds. An odor of butyric acid is
developed.

Cultures in litmus milk show these shreds plastered against the sides of the tube
and showing a pink color.

It is the cause of "foamy organs" occasionally present at autopsy.

88

STUDY AND IDENTIFICATION OF BACTERIA

The best method of diagnosis is to inoculate the culture or material

into the ear vein of a rabbit, kill it and
then incubate the body at 37°C. Gas is
generated in the organs in a few hours.
While the organism is pathogenic for
guinea-pigs it has little effect on rabbits.
B. Perfringens. — This is the name fre-
quently given to the organism which has
assumed such great importance on ac-
count of its causing the gas gangrene so
frequently observed in shell wounds re-
ceived in Belgium. It is considered to
be identical with B. aerogenes capsulatus
(Welch's gas bacillus) and B. phlegm onis
empysematosa. It is very abundant in
the soil of highly fertilized areas.

In size the bacillus is large, 6X 2.5 microns with
square cut ends. It is strongly Gram-positive,
may or may not show a capsule and is nonmotile.
When sporing the spore occurs toward one end
with slight bulging of the rod. Grown in pure
culture spores are rarely found, but in symbiosis
with staphylococci they form abundantly.
Cultures from the clothing of men in the trenches
almost always show the Welch bacillus and less
frequently the tetanus bacillus. Streptococci
were rather frequent. When a shell wound oc-
curs we almost invariably have a gas bacillus
infection which during the first few days gives
rise to a foul-smelling reddish-brown discharge.
Smears from gas gangrene wounds, showing such
discharge, have chiefly the gas bacillus and
streptococci. In the second week the pus be-
comes more purulent and the gas bacillus is infre-
quent. Streptococci, staphylococci and coliform
bacilli are abundant. Glucose agar to which
about Ho c.c. of blood has been added makes
a very favorable medium for the gas bacillus.
It also grows well on milk or blood-serum.
Fleming prefers neutral red egg medium for its
culturing. Cultures of the gas bacillus from gas

gangrene discharges when injected subcutaneously into guinea-pigs kill within
twenty-four hours causing emphysematous swellings. Chlorinated solutions seem

FIG. 25. — B. aerogenes capsu-
latus agar culture showing gas
formation. (Mac Neal.)

THE GAS BACILLUS 89

to be more efficient against the infection than the formerly recommended hydrogen
dioxide.

Achalme isolated a large bacillus from a fatal case of rheumatism
which is now considered as having no relation to acute rheumatism and
which was probably B. aero genes capsulatus.

Kendall has called attention to the importance of this organism in a
certain proportion of cases of summer diarrhoea of infants. (See under
chapter on Faeces.)

CHAPTER VII

STUDY AND IDENTIFICATION OF BACTERIA. MYCO-

BACTERIA AND CORYNEBACTERIA. KEY

AND NOTES

Key for Bacilli. — Having branching characteristics, as shown by par-
allelism, branching, curving forms, V-shapes, clubbing at ends, seg-
mental staining, etc.

J Cultures more or less wrinkled and dry.
Acid-fast. Mycobactenum. < _. ... . ,

[ More like moulds.

I. Grow rapidly on ordinary media at room temperature.
Examples: Timothy grass bacillus of Moeller (B. phlei).

Mist bacillus. Butter bacilli as reported by (i) Rabinowitsch
and (2) Petri.

II. Only grow at about incubator temperature. Scanty growth or none at all
on ordinary media. Media of preference are: (a) solidified blood-serum,
(b) glycerine agar, (c) glycerine potato and (d) egg media.

1. Cultures fairly moist, luxuriant, and flat. Op. temp. 43°C.
a. Bacillus of avian tuberculosis.

2. Cultures scanty, wrinkled, and dry. Appear in ten to fourteen days. Op.
temp. 38°C. Bacilli longer, narrower, more regular in outline and staining
than bovine; vacuolation more marked (2.5/i). Smear from organs of inocu-
lated guinea-pig shows few bacilli. Less virulent for rabbits.

a. Bacillus of human tuberculosis.

Cultures as above, but even more scanty. Bacilli shorter, thicker, less vacuo-
lated (I.SM). Smear from organs of guinea-pig shows many bacilli.

b. Bovine tubercle bacilli.

3. Very difficult to cultivate (Czaplewski).
Smegma bacilli for various animals.

III. Noncul livable by ordinary methods. Cultivable in symbiosis with amoebae.
(Clegg.) Duval cultivated an acid-fast bacillus on N.N.N. medium con-
taining i% glycerine. Bayon cultivated on placental juice glycerine agar a
slightly acid-fast diphtheroid which changed to acid-fast in peritoneum of
mouse. Bayon's organism thought to be similar to Kedrowsky's diphtheroid
of leprosy.

i. B. leprae. Found chiefly in nasal mucus and in juice from lepra tubercles.
Less often in nerve leprosy.

90

NON-PATHOGENIC ACID-FAST ORGANISMS

Nonacid-fast. Corynebacterium. TC°lonies m°r e flat and moist

( Like other bacteria.

I. Do not stain by Gram's method.

i. B. mallei (Glanders). Characteristic culture is that on potato. Growth
like layer of honey by third day. Becomes darker in color, until on eighth
day is reddish brown or opaque with greenish-yellow margin.
II. Gram-positive.

1. Very luxuriant growth on ordinary media. Colonies often yellow to brown-
ish. B. pseudodiphtheriae. Shorter, thicker and stain uniformly.

2. Moderate growth on ordinary media. B. diphtherias. Best media are blood-
serum (Loffler's) or glycerine agar. Has metachromatic granules at poles.

3. Scanty and slow growth on nutrient media. B. xerosis.

THE GROUP OF ACID-FAST BRANCHING BACILLI

There is a large and ever-increasing number of organisms which
have the same staining reactions as the tubercle bacilli, but which differ
in four important essentials of:

1. Growing readily on any media.

2. Showing more or less abundant growth or colonies in twenty-four
hours.

3. Having no pathogenic power for guinea-pigs when inoculated sub-
cutaneously.

4. Not requiring body temperature for development, but growing at
room temperature.

Many of these organisms, if injected intraperitoneally into guinea-pigs will pro-
duce a peritonitis with false membrane. Some also produce granulation tissue
nodules which may be confused with true tubercles. For this reason it is well to
study the lesions in experimental tuberculosis in the guinea-pig. Injected subcuta-
neously, on either or both sides of the posterior abdomen with the needle pointing
toward the inguinal glands, we may have caseation and ulceration at the site of
inoculation. The glands in relation enlarge and caseate. Smears from these show
T. B. The marked and characteristic change is the enormous enlargement of the
spleen, which is studded with grayish and yellow tubercles. Make smears and cul-
tures from the spleen. The death of the guinea-pig usually occurs in about two
months. The lesions may be looked for at three to five weeks.

These nonpathogenic acid-fast bacilli are. of greatest importance by reason of
their possible confusion with the true tubercle bacilli. Their colonies correspond
more or less with different types of tubercle bacilli colonies, being either dry and
wrinkled like human, or moist and irregularly flat as avian. Eventually the moist
colonies become dry and wrinkled. They have been isolated from:

1. Butter and milk.

2. From grasses, especially in timothy grass infusion.

3. In various excretions of animals, as in dung, urine, etc.

4. Normally in man — from skin, nasal mucus, cerumen, and tonsillar exudate.

STUDY AND IDENTIFICATION OF BACTERIA

It is important to remember that such organisms
have very rarely been reported from pulmonary le-
sions, and when present they have been considered
as probably causative.

The present view is that the finding of tubercle
bacilli in sputum has practically as great value as it
had before we knew of these various acid-fast bacteria.

Tubercle Bacillus (Koch, 1882).— This is
a rather long, narrow rod, 3X0.3^1. In the
human type it tends to show a beaded ap-
pearance, this not being due to spores, how*
ever. In the bovine type the staining is more
solid, the organism shorter and thicker, and
shows even a more scanty growth than human
T. B. It has been established that many of
the tuberculous affections of man, especially
those of the skin, bone, and mesenteric glands,
.are of the bovine type, while, as a rule, pul-
monary and laryngeal lesions are of the hu-
man type. Experiments by various com-
missions in different countries have shown
that human and bovine types are very closely
related and that not only may a bovine strain
affect man, but that human T. B. may infect
young calves. As bacilli of the bovine type
have frequently been reported in intestinal
and mesenteric tuberculosis of children it
shows the importance of sterilizing cows'
milk. Koch considered human infection from
bovine sources as of very rare occurrence.

Although Kossel has found only two cases of
bovine T. B. in 709 cases of pulmonary tuberculosis
yet for the other types the findings are different.
Leaving out of consideration the frequency of in-
fections with bovine T. B. in children, recent sta-
tistics have shown that in adults about 4% of cervi-
cal adenitis, 22% of tabes mesenterica and 3. 5% of
bone and joint tuberculosis are due to bovine strains of T. B.

Park and Krumweide, in a study of more than 1000 cases, found
about 10% due to bovine tuberculosis. Of 686 adult cases only 1.3%

FIG. 26. — Bacillus tuber-
culosis; glycerine agar-agar
culture, several months old.
(Curtis.)

TYPES OF TUBERCLE BACILLI 93

showed bovine strains while 352 cases under sixteen showed approxi-
mately 25% of bovine infection. Of 592 cases of pulmonary tuberculosis,
in children and adults, not a single case could surely be regarded as
bovine.

A subject of great moment is that of the atrium of infection in tuberculosis.
While 75% or more of human cases are of the respiratory tract, yet we now have
views that Cornet's idea of T. B. containing dust being aspirated, or Fliigge's spray
method of infection from droplets of sputum in coughing, may be but rarely operative.
The path from intestinal tract to thoracic duct and lungs is direct, so that tuber-
culous bronchial glands of lung infection may be by way of intestines. Some
European statistics, using von Pirquet's method, have shown 90% of children
under fourteen infected while similar American ones have shown about 50%. The
prevailing idea is that we get our infection in childhood and show the disease as we
approach adult life. Infection after adult life is exceedingly rare, as shown by the
rarity of the disease in attendants at institutions for the tuberculous. The
respiratory atria would be just as operative for adults as children so the probable
explanation is that children in crawling about, where tuberculous material is
accessible, may contaminate the fingers and have infection take place by the digestive
tract. With bovine infections we are sure that infection of man takes place almost
exclusively by the alimentary tract.

The British Royal Commission in its final report of July, 1911, considered three
types of T. B.

I. The bovine type belonging to the natural tuberculosis of cattle.
II. The human type. The type more generally found in man.

III. The avian type, belonging to natural tuberculosis of fowls.

The bovine type grows slowly on serum and at the end of two to three weeks
shows only a thin grayish uniform growth which is not wrinkled and not pigmented.
The human type grows more rapidly and tends to become wrinkled and pigmented.
Subcutaneous inoculation of 50 mg. of culture into the neck of calves produced
generalized tuberculosis. A similar injection of human T. B. does not cause general-
ized tuberculosis but only an encapsulated local lesion.

Intravenous injection of o.oi to o.i mg. of bovine T. B. into rabbits causes general
miliary tuberculosis and death within five weeks. With human T. B. in doses of
o.i to i.o mg., similarly injected, the majority of rabbits live for three months.

Subcutaneous injection of 10 mg. bovine T. B. causes death in 28 to 101 days.
Similar injection of human T. B. in doses up to 100 mg. did not kill the rabbits after
periods of from 94 to 725 days. The duration of life in injected guinea-pigs is
longer with human than with bovine T. B.

Subcutaneous injections of bovine T. B. into cats produces generalized tubercu-
losis while the cat is resistant to human T. B. thus given.

Recent statistics (Beitzke) show tuberculous lesions in 58% of adults
at autopsy — Naegli's figures were about 90%.

It is a question whether the avian type is absolutely distinct; many
experiments having indicated the impossibility of infecting fowls with

94 STUDY AND IDENTIFICATION OF BACTERIA

human T. B. Nocard, by inserting collodion sacs containing bouillon
suspensions of human T. B., claims to have changed these to the avian
type. The avian type grows at 43°C. fairly luxuriantly, as a moist,
more or less spreading culture. It grows much better on glycerinated
agar than on serum. Morphologically they are like the human type,
but show less tendency to form compact masses. Very pleomorphic.
Have been reported from sputum of man (doubtful).

Fowls become infected by intravenous or subcutaneous injection or as the result
of feeding. After feeding the lesions are chiefly of the alimentary tract; after in-
jections, of spleen, liver and lungs. Avian T. B. is more virulent for rabbits than
human T. B. but less so than bovine T. B. The mouse is the only animal besides the
rabbit in which avain T. B. can cause a generalized tuberculosis. The conclusions
are that there is no danger to man from avian T. B. With the bovine type it is
quite different as nearly one-half of the deaths in young children from abdominal
tuberculosis were due to bovine T. B. and to that type alone. Not only in children,
but in adolescents suffering from cervical gland tuberculosis, a large proportion were
caused by bovine types. The bovine type is also an important factor in lupus.

There is also a fish tuberculosis. This organism grows much more
rapidly than the other types (three to four days), and grows best at
24°C., growth ceasing at 36°C. The colonies are round and moist.

It is certain that many of the symptoms usually noted in the tuberculous are due
to secondary infections. Pettit, by careful blood cultures, obtained the Pneumo-
coccus in 24 cases and the Streptococcus in 36 cases out of 130 cases studied. He
used from 5 to 20 c.c. of blood from the vein. Positive blood cultures were obtained
in 68% of far-advanced cases, 45% of advanced cases and 16% of incipient cases.

The best culture medium for primary cultures is blood-serum or,
better, a mixture of yolk of egg and glycerine agar. Petroff 's medium
and Dorsett's egg medium are also used. In subcultures, either glycer-
ine agar, glycerine potato, or glycerine bouillon make good media.

In inoculating media from tuberculous material, as, say, from a tuberculous gland
or, more practically, from the spleen of a guinea-pig, the material must be thoroughly
disintegrated or rubbed on the surface of the media so that individual bacilli may
rest on the surface of the culture media. In growing in flasks in glycerine bouillon
a surface growth is desired. The cylindrical flask of Koch gives a better support
to the pellicle than an Erlenmeyer one. In inoculating, a scale of such a surface
growth or a grain from the growth on a slant should be deposited on the surface of
the glycerine bouillon in the flask. In cultivating from sputum use Petroff's
medium.

Inasmuch as the filtrate from cultures has little toxic effect, the
poison is assumed to be intracellular.

DIAGNOSTIC TESTS FOR TUBERCULOSIS 95

Koch's "Old Tuberculin," which was simply a concentrated 5% glycerine bouillon
culture, is now principally used in diagnosis. It was prepared as follows:

After four to six weeks the surface growth begins to sink to the bottom of the
flask. This fully developed culture is evaporated over a water-bath at 8o°C. to
one-tenth the original volume. It is then filtered, the final product containing
about 40% of glycerine.

Koch's "New Tuberculin" or tuberculin "R" was introduced in 1897. In this,
virulent bacilli are dried in vacua and ground up until stained smears fail to show
intact bacilli. One gram of such material is triturated with 100 c.c. water and
centrifugalized. The supernatant fluid is removed and is designated "T. O."
The residue is then dried, triturated in water and centrifugalized. Subsequent
trituration and centrifugalization, preserving each time the supernatant suspension,
gives the new tuberculin. It has been found at times to contain virulent T. B.

Koch's bazillen emulsion has been more recently introduced by Koch (1901).

This is simply a suspension of ground-up bacilli in 50% glycerine solution.

It really is "T. O" and "T. R" combined and contains 5 mg. of bacillary
substance in i c.c. Another preparation is the bouillon filtrate of Denys. This is
the unheated Chamberland filtrate of broth cultures of human T. B. It contains
Y±% phenol.

In the use of T. R. and of bazillen emulsion, Sir A. Wright recommends doses
of Kooo nig., and he rarely goes beyond Kooo rng. in treatment. These prod-
ucts come in i c.c. bottles containing 5 mg. of bacillary material. It is convenient
to remove %o c-c., containing i mg. Add this to 10 c.c. of glycerine salt solution
with %% of lysol. Each c.c. contains Ko nig. One c.c. of this stock solution
added to 99 c.c. of salt solution, with Y±% of lysol, would give a working solution,
each c.c. of which would contain Mo 00 mS- of tuberculin.

For diagnostic tuberculin reactions we have the following:

1. Subcutaneous injection of J£ mg. If no reaction occurs in four
or five days we may increase to i to 5 mg.

Positive reactions show (a) constitutional symptoms of fever, malaise and possibly
chill; (6) focal symptoms, as when a tuberculous gland, joint or skin involvement
becomes active, and (c) local reaction as shown by the tenderness, induration or
inflammation at the site of injection.

2. Variations in opsonic index.

3. Instillation into one eye of a drop of J£% or i % solution of puri-
fied tuberculin.

Reaction is shown by redness, especially of inner canthus, in twelve to twenty-
four hours (Calmette). A previous instillation may sensitize a nontuberculous case
and a second application of the drop may give an erroneous diagnosis. This test
should not be used in persons over fifty or when there is any disease of the eye to be
used or for that matter of the other eye. For instance, in corneal opacities, due to
T. B. keratitis, a focal reaction would occur.

96 STUDY AND IDENTIFICATION OF BACTERIA

4. The cutaneous inoculation method (similar to ordinary vaccina-
tion methods). Scarify two small areas on the arm (J^o mcn m diame-
ter), about 2 inches apart. Rub in one a drop of old tuberculin, in
the other a drop of 25% tuberculin. As a control scarify a spot mid-
way and to one side of the others and rub in i drop of 0.5% carbolic
glycerine. The appearance of bright red papules in twenty-four hours
indicates reaction (von Pirquet).

It is now recommended to deposit a loopful of undiluted tuberculin on the skin
and below that a loopful of saline. A linear incision with a sharp scalpel or glass
needle is made through the saline and then through the drop of tuberculin trying not
to draw blood. After two to five minutes the tuberculin is wiped off. No dressing is
required. Reaction usually appears as induration or inflammatory areola or vesicles
after twenty-four hours, but may be delayed forty-eight hours. This is the method
of preference.

5. Intracutaneous inoculation of i drop of a i-iooo, i-ioo or i-io
dilution of old tuberculin (Mantoux and Moussu).

Webb recommends hypodermic needle points which have been dipped in old
tuberculin and the points allowed to dry. A drop of water is placed on the skin
and the needle points having been moistened in it are plunged through the skin and
withdrawn with a twist. A definite lump shows a positive reaction.

6. Ointment tuberculin test. Rub in 50% ointment of tuberculin
in lanolin. Reaction is shown by dermatitis with reddened papules
in twenty-four to forty-eight hours (Moro).

7. Inoculation of bovine and human tuberculin to diagnose type of
infection (Detre). Of questionable value.

Ebright injects the suspected material into the subcutaneous tissue of one side
of the abdomen of 3 guinea-pigs. At the end of one week an injection into the
other side of the abdomen of one of the guinea-pigs of H c.c. tuberculin is given.
Twenty-four hours later smears are made from the original site of inoculation and
examined for tubercle bacilli. If negative this is repeated with a second guinea-
pig at the end of the second week and finally at the end of the third week with the
third guinea-pig.

Bloch's method is to damage the lymphatic glands in the inguinal region by
squeezing the tissue between the fingers. Injections made there of tuberculosis
material show abundant tubercle bacilli in these damaged glands in ten to twelve
days.

In staining it is better to use the Ziehl-Neelsen method, decolorizing
with 3% hydrochloric acid in 95% alcohol. The alcohol, for all prac-
tical purposes, enables us to eliminate the smegma and similar bacilli,
these being decolorized by such treatment. There are two objections

LEPROSY 97

|

to the Gabbett method, where decolorizer and counterstain are com-
bined: i. We cannot judge of the degree of decolorization — we are
working in the dark; and 2. the matter of elimination of smegma bacilli
is impossible.

Pappenheim's method, in which corallin and methylene blue are dissolved in
alcohol, does not appear to have an advantage over acid alcohol. As a practical
point when the question of tuberculosis of the genito-urinary tract is involved,
inoculate a guinea-pig with urinary sediment.

It must be remembered that in young cultures of tubercle bacilli many of the
rods are nonacid-fast, taking the blue of the counter stain, while older rods are
acid-fast. This frequently causes suspicion of a contaminated culture.

Discussion has arisen as to the granules of Much. These are considered by Much
as resistant forms while others consider them degeneration forms of tubercle bacilli.
At any rate material containing only these Gram-positive granules and no acid-fast
rods may when injected into animals give rise to tuberculosis and acid-fast bacilli.

The combination of the acid-fast and Gram-staining methods as recommended
by Fontes is very satisfactory.

Bacillus Leprae (Hansen, 1874). — This is the cause of leprosy. In
nodular leprosy the organism is readily and in the greatest abundance
found in the juice of the tubercles of the skin, and secretions of ulcera-
tions of nasal and pharyngeal mucosa.

The earliest lesion is probably a nasal ulcer at the junction of the bony and
cartilaginous septum. Scrapings from this ulcer may give an early diagnosis.

In the skin they are chiefly found in the derma packed in the so-called lepra
cells. The process is granulomatous but does not show the caseation of tubercu-
losis or the predominant plasma cells of syphilis. The bacilli are also found engulfed
in the endothelial cells lining the lymphatics.

They are also found in the glands in relation to the superficial lesions. The
bacilli are found in smaller numbers in the liver and spleen. In anaesthetic or nerve
leprosy they are found in small numbers in the granuloma tissue which affects the
interstitial connective tissue of the peripheral nerves. Also, rarely, in the anaesthetic
spots of nerve leprosy.

The leprosy bacilli are found in profusion in the granulomatous tissue of the
corium and subcutaneous structures of the leprous nodules, chiefly within cells
called "lepra cells" and also within endothelial and connective-tissue cells as well
as lying free, packed in lymphatic channels, the so-called "globi."

The leprosy bacillus may be distinguished from the tubercle bacillus
by the following points:

i. The presence ordinarily of huge numbers of bacilli often grouped
in packets like a bundle of cigars tied together. It will be remembered
that it is very difficult to find even a single tubercle bacillus in a skin
lesion. Leprosy bacilli form palisade groups but not chains.

98 STUDY AND IDENTIFICATION OF BACTERIA

•#•

2. The leprosy bacilli stain more solidly and when granules are pres-
ent they are coarser and more widely separated than the fine granula-
tions of the tubercle bacillus.

3. They do not stand decolorization quite as well as the tubercle
bacillus. With 20% sulphuric acid in water they hold their color almost
as well as tubercle bacilli but with 3% HCL in alcohol they decolorize
in about two hours as against twelve to twenty-four hours for the
tubercle bacillus.

4. Leprosy bacilli have neither been surely cultivated nor surely
inoculated with pathogenic results into guinea-pigs or other experi-
mental animals and it is by the negative results upon cultivating or
animal inoculation that we have our surest method of differentiation
from tubercle bacilli.

Leprosy bacilli are chiefly spread through the lymphatics, but in nodular leprosy,
their occurrence in the blood stream during the febrile accessions is so constant that
this route may also be of importance. Next to the corium they are most abundant
in the lymphatic glands. They stain readily by Gram's method.

A great amount of work has been done within recent years in attempting to
cultivate the leprosy bacillus.

In 1900 Kedrowsky, culturing material from 3 cases of leprosy obtained diph-
theroids from 2 and a streptothrix from i. A rabbit was inoculated first in-
travenously and later intraperitoneally with this nonacid-fast streptothrix and
when killed six months later showed peritoneal nodules from which both diphtheroids
and acid-fast bacilli, but not a streptothrix, were recovered culturally. Injection of
cultures of the acid-fast bacilli and diphtheroids into rabbits and mice produced
nodules which, when cultured, showed acid-fast organism or diphtheroids. In
1901, he cultivated a diphtheroid from a fourth case of leprosy.

Fraser and Fletcher working with Kedrowsky's culture produced peritoneal
nodules with the killed as well as the living organism. They were able to produce
the same results with B . phlei.

With emulsions of leprous nodules, rich in leprosy bacilli, they could not produce
similar lesions in the experimental guinea-pigs.

Rost obtained a culture on a salt-free medium from which he prepared his leprolin
by a process similar to that used for old tuberculin. It was claimed that leprolin
had marked curvative power in leprosy. Recently Williams and Rost have culti-
vated a streptothrix on a medium containing milk.

Clegg, by inoculating his medium with cultural amoebae, obtained growth of
diphtheroid organism, with acid-fast tendencies, from the spleen pulp of lepers.

Duval, by using media containing amino-acids, as result of tryptic digestion,
brought forward two organisms, one of which was a diphtheroid and grew luxuriantly
while the other showed a slow scanty growth and was acid-fast.

Bayon, by using placental media, isolated an organism rather resembling that
of Kedrowsky. These organisms alone responded to immunity tests when si

DIAGNOSIS OF LEPROSY 99

were made by Bayon and they alone gave rise to tissue changes resembling those of
leprosy when injected into animals.

Professor Deycke obtained a streptothrix-like growth from the granulomatous
tissue of excised leprous nodules. The ethereal extract from this culture gave a
neutral fat which he called nastin and which is the basis of a leprosy treatment.

Quite recently and after working for eighteen months, with material
from 32 nonulcerative cases of nodular leprosy, not only with media as
recommended by Duval, Rost and Bayon, but with blood and serum
culture media, both by aerobic and anaerobic procedures, Fraser has
been unable, in a single instance, to obtain any evidence of growth from
this wealth of leprosy material.

As being opposed to the possibility of culturing the human leprosy bacillus, it
may be stated that most of the experiments along this line with rat .leprosy, a
disease occurring naturally in rats and caused by an organism almost identical, as
to lesions produced, with the leprosy bacillus, have been negative. Bayon, however,
states that he has cultivated the bacillus of rat leprosy.

Recently a leprosy-like disease of rats has been reported in which there are two
types: i. A skin affection and 2. a glandular one. In this disease, acid-fast,
bacilli, alike in all respects to leprosy bacilli, have been found. Deane has obtained
a diphtheroid-like organism in culture, which is nonacid-fast. This same finding
has been obtained in cultures considered positive in human leprosy.

There have been many reports of positive findings with the Wassermann test in
cases of tubercular leprosy but such reports are considered doubtful by many.
Butler, in the Philippines, has found that the lepers gave no higher percentage of
positive Wassermann reactions than did the nonleprous native patients at his
clinic.

There is nothing definitely known as to method of transmission of
the disease.

In rat leprosy it has been found that infection of other rats takes place as readily
through slight abrasions of the skin as when material is injected subcutaneously.

The idea is that natural infection occurs by way of the skin and through the
lymphatics. There is no evidence that insects play a part in transmission.

Rat leprosy prevails extensively in Europe, Asia and America. Although similar
etiologically and pathologically there does not seem to be any connection between
the disease in rat and in man, as is the case with human and rat plague.

Laboratory Diagnosis,— The usual procedure is to scrape a spot or
nodule with a scalpel until the epidermis has been gone through and
then smear out the serous exudate on a slide and stain by the Ziehl-
Neelsen acid-fast method or by Gram's stain. Twenty percent sul-
phuric acid is less apt to decolorize than the 3% acid alcohol, the lep-
rosy bacilli being less resistant to acid alcohol decolorization than to

100 STUDY AND IDENTIFICATION OF BACTERIA

aqueous acid solutions. There is a great variation in the resistance to
decolorization of leprosy bacilli, a preparation from one case holding its
color almost as well as tubercle bacilli, while material from another
case may decolorize very easily.

ik

I am partial to Tschernogabow's technic. In this one punctures the sub-
epithelial granulomatous tissue with a capillary pipette, the end of which has been
broken off by tapping the point in order to give a cutting point, and the serum which
exudes is smeared out and stained.

Some prefer emulsifying a piece of the tissue and centrifuging and staining the
sediment. Quite recently the antiformin method of treating leprous tissue, as for
tuberculous tissue, has been used.

Many insist that the best method is to cut out small sections of the lesion, going
well into normal tissue, and putting through paraffin and cutting thin sections and
staining. Gram's method, counterstaining with Bismark brown gives beautiful
preparations. For acid-fast staining first stain with haematoxylin to obtain a his-
tological background and then steam with carbol-fuchsin, decolorize very briefly
with acid alcohol, then through absolute alcohol and xylol.

Of the greatest diagnostic value is the staining of the nasal mucus
or scrapings from ulcerations on nasal septum for leprosy bacilli.
These are often found in the characteristic cigar package bundles or
engulfed in lepra cells. A standard procedure is to give 60 grains of
iodide of potash to cause a drug coryza, in the secretions of which lep-
rosy bacilli may be found. However, one will have better success if
the nasal secretion be obtained at a time when a natural coryza exists.

Thibault examined the nasal mucus, gland juice and blood of 30 lepers. He
obtained leprosy bacilli in the nasal mucus of 20, in the gland puncture juice of 18,
and in the blood of 7.

Hollman detected leprosy bacilli in the nasal mucus of 90% of 58 nodular
cases, of 67% of 6 mixed leprosy and of 45% of anaesthetic cases, after making 329
examinations.

Leprosy bacilli are apt to be found in the blood of nodular cases, especially at the
time of the febrile accessions. The blood is best taken in 5 or 10 c.c. quantities into
i% sodium citrate in distilled water. After centrifuging, the sediment is treated
with 10% antiformin, at 37°C. for one hour. Again centrifuging, and washing, the
sediment is smeared out on a slide and stained. The bacilli are not apt to be found
in the blood of cases of nerve leprosy.

Gland puncture has recently been considered as an important diagnostic procedure
in leprosy.

It must not be forgotten that while the finding of leprosy bacilli is
usually very easy in the nodules of nodular leprosy it is a painstaking
and discouraging procedure with the spots of nerve leprosy. Even the

GLANDERS IOI

affected nerves, at autopsy, often fail to show bacilli. For nerve-lep-
rosy the examination of nasal mucus is of prime importance.

The X-ray has been utilized in the recognition of the very early, trophic changes in
bone, showing the commencing absorption of phalanges.

NONACID-FAST BRANCHING BACILLI

Bacillus Mallei (Loffler and Shutz, 1882).— This is the cause of a
rather common disease of horses. When affecting the superficial
lymphatic glands, it is termed "farcy;" when producing ulceration of
nasal mucous membrane, the term "glanders" is used.

In man there are 2 types of glanders — chronic and acute. In the
chronic form an abrasion becomes infected from contact with glanders
material and an intractable foul discharging ulceration results. This
may persist for months with lymphatic involvement or may become
acute. The acute form may also develop from the start and the cases
are usually diagnosed as pyaemia. There is great prostration with
marked pains of the extremities. Death invariably results in acute
glanders. The bacillus is a narrow, slightly curved rod, about 3X0.3^1.
It is nonmotile and Gram-negative. It at times presents a beaded ap-
pearance. In subculture on agar or blood-serum the growth is some-
what like typhoid but more translucent. In original cultures from pus
or tissues the colonies may not show themselves for forty-eight hours.

As the organism does not tend to invade the blood stream, blood
cultures are apt to be negative. The glanders bacillus grows best on
an acid glycerine agar (+2).

The characterisitc culture is that on potato. Grown at 37°C., we
have a light brown or yellowish honey-like or mucilaginous growth,
which by the end of a week spreads out and takes a cuprous oxide like
reddish tint with greenish borders. The potato assumes a dirty brown
color. This and the inoculation of a guinea-pig are the chief diagnostic
measures. If the material is injected intraperitoneally into a male
guinea-pig, marked swelling of the testicles is noted within forty-eight
hours, at the earliest, to seven to ten days. Cultures should be made
from this swollen testicle as other organisms than glanders may bring
it about.

Only the B. pyrocyaneus and cholera vibrios give a similar coloration of potato
These organisms, however, are easily differentiated. The glanders bacillus is the
most dangerous of laboratory cultures and should be handled with extreme care.

102

STUDY AND IDENTIFICATION OF BACTERIA

The best stains are carbol thionin and formol fuchsin. In sections stained with
carbol thionin the bacilli are apt to be decolorized by the subsequent passage of
the section through alcohol and xylol. This may be avoided by blotting carefully
after the thionin, then clearing with xylol or some oil and mounting. Nicolle's
tannin method is a good one.

Mallein is prepared by sterilizing cultures that have grown in glycerine bouillon
for about a month by means of heat (ioo°C.). The dead culture is then filtered
through a Berkefeld filter and the filtrate constitutes mallein. It is chiefly used as
a means of diagnosing the disease in horses. The reaction consists in rise of tempera-
ture and local oedema. The dose is about i c.c.

Agglutination and complement-fixation tests are also used for diagnosing glanders.

Bacillus Diphtheria (Klebs discovered, 1883 ; Loffler cultivated, 1884).
— The diphtheria bacillus is found not only in the false membrane which

FIG. 27. — Bacillus of diphtheria. (Xiooo.) (Mac Ned.)

is so characteristic of the disease, but may be found in abundance in
the more or less abundant secretions of nose and pharynx. In studying
the epidemiology of diphtheria, especial attention must be given to the
examination of nasal discharges.

With diphtheria carriers it is important to remember that the crypts of the tonsils
may harbor the bacilli and thus protect them from the ordinary application of
antiseptic agents. Goldberger found that the combination of throat and nose cul-
tures gave much higher findings with carriers than either separately. The nose
cultures gave more positives than the throat ones. These workers only obtained
about i % of positives in 4093 cases in Detroit, these being lower than figures from

DIPHTHERIA

I03

other sources. It is interesting to note that 32% of these people showed pseudo-
diphtheria bacilli.

Infection of the larynx and middle ear are not very rare. The mu-
cous membrane of the vagina or the conjunctiva may also be infected.
The B. diphtheria may be in pure culture lying entangled in the fibrin
meshes or contained within leukocytes in the membrane or be asso-
ciated with staphylococci, pneumococci, or especially streptococci.
These latter complicate unfavorably and cause the suppurative con-
ditions about the neck. In fatal cases the diphtheria bacillus may be
found in the lungs. Ordinarily, however, it remains entirely local and
does not get into the circulation or viscera.

FIG. 28. — Diphtheria bacilli involution forms. (Kolle and Wassermann.)

It produces soluble absorbable poisons which are designated toxin in the case
of the one responsible for the acute intoxication, parenchymatous degeneration and
death and toxone for the poison which produces oedema at the site of inoculation and
postdiphtheritic palsy. The injection of the soluble poisons alone without the bacilli
produces the symptoms of the disease.

The bacilli tend to appear as slightly curved rods, showing varying
irregularities in staining, as banding or beading, and in particular the
presence at either end of small, deeply staining dots (metachromatic
granules).

These granules may be seen in an eighteen-hour culture, but are more abundant
after thirty-six hours. The granules are well seen with Loffler's blue, but better
with Neisser's method. In culture the bacilli show swelling at one or both ends or
clubbing. In secretions or in culture they show V-shapes or false branching and,
what is most characteristic, the parallelism— four or five bacilli lying side by side like

104

STUDY AND IDENTIFICATION OP BACTERIA

palisades. Being a Gram-positive organism while the majority of the other patho-
genic bacilli are Gram-negative, it is of greatest importance to stain smears by this
method. It is not so strongly tenacious of the gentian violet as the cocci, so decolori-
zation should not be carried too far.

The best medium for growing it is Loffler's blood-serum.

An egg medium, made of the whole egg with glucose bouillon as described pre-
viously, is as suitable as Loffler's serum. Coagulated white of egg answers fairly
well, as will a hard-boiled egg — the shell at one end being cracked and the white cut
with a sterile knife. This smooth side is then inoculated and the egg placed cut
side downward in a sherry glass. If an incubator is not at hand a tube may be
carried next the body in a pocket. The bacillus grows better on glycerine agar than
on plain agar. On such plates they appear as small, coarsely granular colonies with
a central dark area. In size the colonies resemble the streptococcus. On blood-
serum the' colonies are larger — ^2 to ^ inch in diameter.

FIG. 29. — B. diphtheria stained by Neisser's method. (Mac Ned.}

The diphtheria bacillus grows luxuriantly on blood agar and like the Streptococcus
pyogenes has a yellowish laked zone around the colony. The Hofmann and the
xerosis bacillus do not seem to have this haemolytic power. In bouillon it tends
to form a surface growth. It is at the surface that the toxin function is most marked,
hence in growing diphtheria for toxin formation we use Fernbach flasks which expose
a large surface to the air. It is a marked acid producer — bouillon with a -f- 1 reac-
tion becoming +2.5 to +3 in thirty-six hours. The nitrate from a two or three
weeks' old broth culture is highly toxic, and is usually referred to as diphtheria toxin.
It is used in injecting horses to produce antitoxin. Ehrlich uses as a standard to
measure the toxicity of toxin the minimal lethal dose (M. L. D.). This is the amount
of toxin which will kill a 250-gram guinea-pig in just four days. Some toxins have
been produced whose M. L. D. was ^Oo c.c. or 0.002 so that i c.c. of such toxin would
kill 500 guinea-pigs. Theoretically, the measure of an antitoxin unit is the capacity
of neutralizing 200 units of a pure toxin. (On exposure to light, etc., toxin loses its
toxic power and is termed toxoid.)

DIPHTHERIA TOXIN 105

Almost invariably a bouillon filtrate contains toxones and toxoids besides the toxin.
For all practical purposes it is usual to consider an antitoxin unit as that amount
which will neutralize 100 M. L. D. (theoretically 200 units). Thus it would be
the amount which would neutralize 0.2 c.c. of the above noted toxin. In testing
filtrates as to their toxicity we make use of two limits, one designated Lo and the
other L+. When we add increasing amounts of a filtrate containing toxins and
toxones (the toxoids are less important because they do not show either the local
reaction or palsy of toxones and do not possess the acute death-producing power of
the toxin; they do, however, have combining power for antitoxin and are complicating
factors in standardization) to an antitoxin unit we gradually reach a point where the
slightest further increase will bring about slight reaction at site of inoculation of
the guinea-pig and possibly some slight paralysis. These symptoms are due to
toxone action. The amount of the toxic broth filtrate which is completely neu-
tralized by one antitoxin unit is called Lo. Upon further addition of the filtrate
to this Lo amount we finally reach a point when death of the 25o-gram guinea-pig
occurs in four days. This is called the L+ or fatal dose. Instead of having to add
only that amount of filtrate which is capable of killing the guinea-pig (i M. L. D.),
it is found that we must add an amount sufficient to kill 10 to 20 pigs. The explana-
tion is that while toxin and toxone both have power to combine with antitoxin and
to be neutralized, toxin has greater affinity and can dispossess toxone of its attach-
ment. Consequently when all the combining strength of the antitoxin unit (equal
to 100 M. L. D.) has neutralized the toxins and toxones added to it there is a complete
blocking of injurious action of toxins or toxones. When adding more filtrate to the
fixed amount of antitoxin a toxin molecule dispossesses a toxone molecule of its hold
on the antitoxin unit. The toxin is neutralized but a toxone is put in circulation
and is capable of causing reaction and palsy. Not until every toxone molecule has
been displaced can the further addition of i M. L. D., containing sufficient toxin to
kill, be free from the neutralizing antitoxin and capable of producing death in a
250-gram guinea-pig in four days. There have been prepared at the Hygienic
Laboratory in the U. S. and in various European laboratories, by laborious testing
of antitoxic serum, standardized antitoxin. By keeping dried antitoxic serum in a
vacuum tube under conditions preventing exposure to heat, light and moisture the
strength of the antitoxin unit remains stable. It may be stated that standardized
toxin soon tends to change in potency, therefore it is customary to take exactly
i unit of antitoxin and by adding to it increasing amounts of toxin to determine the
amount which will be sufiicient to kill the guinea-pig in four days. This is usually
somewhat over the amount which will kill 100 guinea-pigs, or 100 M. L. D., and is
designated the L+ dose of toxin. In practical application at biological product
institutions the L+ dose is tested with increasing amounts of antitoxic serum, as
drawn from the immunized horse, and that amount of serum which when mixed
with the L+ dose of toxin just allows death of the guinea-pig in four days is accepted
as one antitoxin unit.

In the preparation of antitoxin horses are employed; the method being to inject
the bouillon filtrate or toxin subcutaneously at weekly intervals for a period of three
or four months. When each c.c. of the serum of the horse is found to contain about
250 to 500 antitoxin units the horse is bled from the jugular vein. Some sera contain
as much as 1300 units in a cubic centimeter.

Methods of purifying and concentrating antitoxin are now employed by certain

106 STUDY AND IDENTIFICATION OF BACTERIA

makers, the principle being that the antitoxin in the horse serum is precipitated
with the globulins which come down on half saturation with ammonium sulphate.
In this way, as the content in horse-serum proteids is lessened, the anaphylactic
dangers are lessened.

As a curative measure, from 2500 to 5000 units should be injected.
If the injection is delayed or the case very serious the dose should be
10,000 units. As much as 50,000 units has been given in severe cases.
The prophylactic dose is 500 units.

Schick Reaction. — By the employment of this reaction we can under-
stand why one child develops clinical diphtheria and another only shows
the organism in the throat (laboratory diphtheria). We find that cer-
tain persons have sufficient amount of diphtheria antitoxin normally in
the circulation to protect against the soluble toxin elaborated by the
organisms localized in throat or nose. Such cases show either a mini-
mal or negative reaction.

Persons not having any antitoxin in the circulation show a positive reaction.
The test is performed as follows: With a small sharp hypodermic needle we inject
intradermally ^0 °f a minimum lethal dose (i M. L. D.) of diphtheria toxin as
determined for a 25o-gram guinea-pig. The standardized toxin is so diluted with
a ^% carbolic acid solution that o.i c.c. contains ^so of a M. L. D. A positive
reaction shows within twenty-four hours, reaching its maximum intensity in two
days, as a reddened area, about i inch in diameter with more or less induration.

The reaction persists for about a week, leaving a brownish pigmentation. Positive
reactions show that the patient has less than %Q oi a. unit of antitoxin in i c.c. of
his blood-serum and that he possesses no immunity to diphtheria.

This test is of great value as showing the cases needing prophylactic
injections of antitoxin. Furthermore nurses showing a positive reac-
tion should not take care of diphtheria patients. Carriers of true
diphtheria usually show a negative reaction as contrasted with
pseudodiphtheria ones.

It is of value in showing duration and degree of immunity following antitoxin
injections and such investigations have shown that intravenous injections are the
most efficient, next the intramuscular and least efficient the subcutaneous route.
Moody obtained an average 'of 45.2% positives in 524 people examined.

Sudden death after administration of antitoxin has been reported in
cases of status lymphaticus. (See anaphylaxis.)

Laboratory Diagnosis. — In obtaining material from a throat, be sure that an
antiseptic gargle has not been used just prior to taking the throat swab. The part
of the swab which touched the membrane or suspicious spot should come in contact
with the serum slant. This is best accomplished by revolving the swab. An im-

DIPHTHEROIDS Ioy

mediate diagnosis is possible in probably 35% of cases by making a smear from a
piece of membrane. In doing this Neisser's stain or the toluidin blue stain are
usually considered the most satisfactory. I prefer the Gram stain, however. The
diphtheria bacilli found in such smears are not apt to be clubbed and stain more
uniformly.

If there is any doubt about the nature of an organism in a throat culture, always
stain: i. with Loffler's alkaline methylene blue for two minutes; 2. with Gram's
method, being careful not to carry the decolorization too far, and 3. by Neisser's
method. With Loffler's you obtain a picture which, after a little experience, is
characteristic; at times the polar bodies show as intense blue spots in the lighter
blue bacillus. One is liable to confuse cocci lying side by side for diphtheria bacilli
with segmental or banded staining. This difficulty is not apparent when Gram's
staining is used. This gives us great information, as the diphtheria and the pseudo-
diphtheria are the only small Gram-positive bacilli usually found in the mouth.
The cocci are also well brought out. Neisser's stain gives a picture which, when
satisfactory, is almost absolutely characteristic. You have the bright blue dots
lying at either end of the light brownish-yellow rods. When first isolated from a
throat, the diphtheria bacillus is apt to stain characteristically by Neisser. Later
on, in subculture, there may be no staining of the polar bodies. Neisser originally
recommended five seconds' application, with an intermediate washing, for each of
his two solutions. Thirty seconds for each is probably preferable. Some authorities
recommend five to thirty minutes. It is well to bear in mind that about 2% of the
people in apparent health carry diphtheria bacilli of the granular or barred type in
their throats and of these about one in five will prove virulent for the guinea-pig.

It is essential when a question exists as to the nature of a diphtheria-
like organism to test it as to virulence. While there are exceptions,
especially in freshly isolated colonies, yet as a rule a severe infection
yields virulent organisms and vice versa. Pure cultures are best ob-
tained by streaking material from the throat on glycerine agar plates.
From an isolated colony inoculate a tube of bouillon. From such a forty-
eight- or seventy-two-hour-old culture inoculate a guinea-pig with 2 or 3
drops subcutaneously in the shaven abdomen. Escherich considers a
fatal result with 1.5 c.c. of such a bouillon culture a satisfactory test as to
virulence. After death, which occurs in two or three days, the adrenals
are enlarged and haemorrhagic. The diagnosis is more sure if, in addi-
tion to the first animal, a second one, which has had antitoxin, is inocu-
lated. The protected one should live.

Diphtheroid Bacilli. Pseudodiphtheria Bacillus. Hofmann's Bacil-
lus.— Under these terms various Gram-positive bacilli have been de-
scribed as occurring in genito-urinary, nasal and skin diseases.

Their chief importance is in connection with their presence in the
throats of healthy people. Probably approximately 10% of people
harbor such organisms as against i to 2% with granule types. Some

108 STUDY AND IDENTIFICATION OF BACTERIA

authorities believe it possible for these diphtheroids to be capable of
being transformed into virulent diphtheria bacilli. This seems im-
probable. Such organisms are often found in urethral discharges,
either alone, or with gonococci or other organisms.

Recently a great deal of attention has been given to the etiological relationship
between diphtheroids and Hodgkin's disease. Fox, in a critical study of this
relationship, has obtained diphtheroids of varying morphology and cultural charac-
teristics from such glands as well as from enlarged glands of chronic atrophic arthritis
and other conditions. It would appear conservative to reject the acceptance of
diphtheroids as causative agents not only in this disease but in leprosy as well.

Negri has applied the name Coryne bacterium granulatis malignl to diphtheroids
isolated from glands in Hodgkin's disease. The granular rods of Much, supposed
to be connected with tubercle bacilli, may be diphtheroids. Mallory has connected
diphtheroids with scarlet fever.

DIPHTHEROID CHARACTERISTICS

1. They very rarely give the blue dot staining at the two ends. Exceptionally
they may give a dot at one end. Neisser attaches importance to the dots at both
ends as showing diphtheria.

2. They tend to stain solidly or at most with only a single unstained segment.
They are shorter, thicker, and do not curve so gracefully as the true diphtheria
bacillus. They are stockier.

3. They produce very little acid in sugar media, not one-half that produced by
true diphtheria. Goldberger found 29 out of 30 cultures of B. diphtheria virulent
and acid producers. Of 47 Hofmann cultures 6 showed slight acid production while
41 produced alkali. All were nonvirulent.

4. They are nonpathogenic for guinea-pigs.

5. Many of them grow quite luxuriantly and often show chromogenic power.

Xerosis Bacillus. — This organism is frequently found in normal con-
junctival discharges. There is question as to its pathogenesis, and the
finding of this organism should not exclude the previous presence of
strictly pathogenic organisms, such as the Gonococcus or the Koch-
Weeks. It resembles the diphtheria bacillus in being Gram-positive
and showing parallelism, but differs i. in being nonvirulent for guinea-
pigs; 2. in requiring about two days for the appearance of colonies; 3.
in not showing Neisser's granule staining, and 4. in producing very
little acid in sugar media.

CHAPTER VIII

STUDY AND IDENTIFICATION OF BACTERIA. GRAM-
NEGATIVE BACILLI. KEY AND NOTES

KEY to the recognition of nonspore-bearing, nonchromogenic, non-
Gram-staining, nonbranching bacilli.

(NOTE. — Some books say that the proteus group is Gram-positive. It is, how-
ever, usually negative.)
Do not grow on ordinary media. Require blood agar (haemophilia bacteria), serum

agar, or blood-serum.

Minute dewdrop colonies.

1. Influenza bacillus. Requires blood media.

2. Koch- Weeks bacillus (conjunctivitis). Serum agar best medium. Many,
however, regard haemoglobin as necessary for growth.

3. Muller's bacillus of trachoma. Like Koch- Weeks bacillus, but easier to
^cultivate.

4. Morax diplobacillus of conjunctivitis. Grows well and produces little pits
of liquefaction in Loffler's blood-serum.

5. Bordet-Gengou bacillus of whooping-cough. Does not grow on LofBer's
serum. Requires blood or ascitic fluid agar. Original isolation should be on
glycerine potato agar.

6. Ducrey's bacillus (soft chancre). Requires media rich in blood or serum.
Forms chains.

Grow well on ordinary media.

I. Cultures in litmus milk. PINK.

A. Nonmotile.

Lactis aerogenes group. B. lactis aerogenes.

Produce gas in glucose, lactose, and saccharose. No liquefaction of gelatin.
Short, stubby bacteria, often showing capsules. Intermediate between the
colon and Friedlander group.

B. Motile.

1. Nonliquef action of gelatin.

(a) B. coli group. Coagulation of milk. No subsequent peptonization.
Gas in glucose and lactose, none in saccharose. Indol produced.
Neutral red reduced.

2. Liquefaction of gelatin.

(a) B. cloaca? group. Gas in glucose, slight in lactose. Slow coagulation
of milk. Subsequent peptonization.
109

110 STUDY AND IDENTIFICATION OF BACTERIA

II. Cultures in litmus milk. LILAC.
A. Nonmotile bacilli.

1. No gas generated in glucose or lactose bouillon.

(a) Haemorrhagic septicaemia group. These are oval bacilli with tend-
ency to bipolar staining.

Colonies smaller and less opaque than those of B. coli.

Examples: B. pestis, B. suisepticus, B. cholerae gallinarum (chicken

cholera).

B. pseudotuberculosis rodentium (very similar to plague).

B. pestis is absolutely nonmotile, does not liquefy gelatin, does not

produce indol, produces slight acid in glucose but not in lactose

bouillon.

(b) Dysentery group. Colonies similar to those of B. coli.
Divided into two classes according as mannite is acted on :
Those not giving acid — nonacid group — (Shiga-Kruse).
Those giving acid — acid group — (Flexner-Strong).

2. Gas generated in glucose bouillon not in lactose.

(a) Friedlander group. Give very viscid, porcelain-like colonies.
Tendency to capsule formation in favorable media.
Examples: B. pneumonias, B. capsulatus mucosus, B. rhinoscleroma
B. Motile bacilli.

1. Do not liquefy gelatin.

(a) Do not produce gas in either glucose or lactose bouillon.
Typhoid, or Eberth group. No indol. No coagulation of milk. No
reduction of neutral red.

(b) Gas generated in glucose, not in lactose media. Milk not coagulated.
Neutral red reduced.

Gartner group. This includes:

Pathogenic types for man; as B. enteritidis, B. icteroides, B. para-
typhoid B, B. psittacosis. Nonpathogenic for man; as B. cholerae
suum (hog cholera).

2. Liquefy gelatin.

(a) Proteus group. Colonies at first round later amoeboid, spreading.
Produce gas in glucose, not in lactose. Produces foul odor.
B. zopfii type of Proteus group does not liquefy gelatin; colonies at first
round, later amoeboid, spreading. Foul odor in cultures. Gelatin
stab shows lateral branching.

NOTE. — The Friedlander and the lactis aerogenes group, differ culturally chiefly
in carbohydrate fermentation activities and organisms considered as belonging
to the Friedlander group rather than to the lactis aerogenes group may show acid
in litmus milk. Where an organism having the characteristics of B. coli, but fer-
menting saccharose, is found, it is termed B. coli communior. A nongas producing
colon type organism has been designated B. coli anaerogenes. Certain organisms
which turn litmus milk lilac and which liquefy gelatin, but do not produce gas in
sugar media, belong to the "Booker" group. Other organisms which acidify and
coagulate litmus milk but do not liquefy gelatin or produce gas in glucose or lactose
media have been placed in the "Bienstock" group. The proteus or Hauser group is

INFLUENZA III

composed of organisms showing various functions; Prcteus vulgaris liquefying gelatin
rapidly, P. mirabilis slowly and P. zenkeri not at all. The differentiation of the colon
group is extensively considered under bacteriology of water.

GRAM-NEGATIVE BACILLI REQUIRING SPECIAL MEDIA

Bacillus Influenzas (Pfeiffer, 1892). — This organism is the type of the
so-called haemophilia bacteria — organisms whose growth is restricted
to media containing haemoglobin. The influenza bacillus seems to grow
better on slants freshly streaked with blood than on those which have
been made for some time, and they appear to grow better on this sur-
face smear of blood than on a mixture of agar and blood.

The influenza bacilli are most likely to be isolated from the sputum of broncho-
pneumonia due to this organism. It has also frequently been found in the nasal
secretions of influenza patients. Exceptionally, it is present in the blood, and has
been isolated in cases of meningitis from cerebrospinal fluid. It also occurs at times
in anginas, but then usually associated with other organisms. Infection probably
only takes place by contact. It is a very small bacillus which in sputum tends to
show itself in aggregations, especially centering about M. tettragenus. It stains rather
faintly when compared with cocci, so that a smear of sputum stained with formol
fuchsin shows a deep violet staining for the M. tetragenus or other cocci, and scattered
around in a clump-like aggregation we see these minute, rather faintly stained rods.
They also tend to stain more deeply at either end, so that they sometimes appear
as diplococci. Gram's method, counterstaining with formol fuchsin is excellent for
their demonstration. The red bacilli and the violet-black cocci are easily dis-
tinguished .

To cultivate them, rub the sputum, or at autopsy the material from
a lung, on a slant smeared with human blood (pigeon's blood is also
satisfactory), and then without sterilizing the loop, inoculate a second
blood slant; then a third, and possibly a fourth.

The colonies appear as very minute dewdrop-like points which seem to run^into
each other in a wave-like way. To test such colonies we should transfer a single
colony to plain agar and blood-serum, trying not to carry over any blood. If the
least trace of blood is carried over, they may grow on agar or blood-serum. Organ-
isms resembling the influenza bacillus have been isolated from whooping-cough.
Such organisms have also been found in the fauces of well persons. In many
epidemics of influenza the bacillus has not been isolated, or success has obtained in
only a small proportion of the cases. Etiological factors in conditions more or less
resembling influenza may be the Streptococcus, Pneumococcus, or M. calarrhahs.
The influenza bacillus seems to grow best in symbiosis with some other organism,
especially with S. pyogenes aureus.

Koch-Weeks Bacillus (Koch, 1883).— This produces a severe con-
junctivitis. It is very common in Egypt and is also a frequent cause
of conjunctivitis in the Philippines and in temperate climates.

112 STUDY AND IDENTIFICATION OF BACTERIA

Smears made from conjunctival secretion show large numbers of small Gram-
negative bacilli, especially contained within pus cells, but also lying free. They are
more difficult to cultivate than the influenza bacillus, but the same general methods
hold. The vitality of this organism is very slight so that almost immediate trans-
ference of material is necessary. Flies are an important factor in Egypt. The
period of incubation is short, twelve to thirty-six hours. The best medium is a
mixture of glycerine agar and hydrocele or ascites fluid. At first we rarely obtain
pure cultures. The colonies are dewdrop-like and first show themselves in about
thirty-six hours in incubator cultures.

It would seem that blood agar is a better medium than a serum one.
Many haemoglobinophilic bacteria will grow with only i to 500 haemo-
globin, so that growth on serum might be explained by slight blood
admixture.

Some have thought that repeated infection with the Koch- Week's bacillus was
the cause of trachoma. Others have regarded other haemoglobinophilic bacteria as

FIG. 30.— The Koch- Weeks Bacillus. (Hansell and Sweet.)

causative. According to the views of Park and Williams the inclusion bodies of
Prowazek, supposed to be characteristic of trachoma, are simply clumps of extremely
small, coccoid haemoglobinophilic bacteria. Besides these organisms the Gonococcus
in smears from gonorrhceal ophthalmia is stated to show involution forms, making
a resemblance to trachoma bodies.

Noguchi thinks that while there is a morphological similarity between degenerated
haemoglobinophilic bacteria and cell inclusion^, yet in the latter, the elementary
bodies are much smaller than the bacterial granules, and the initial bodies less definite
in contour. He was able to infect the conjunctivae of monkeys with inclusion bodies
material, but not with haemoglobinophilic bacilli.

Diplobacillus of Morax. — This organism causes mild blepharo-con-
junctivitis chiefly at the internal angle of the eye. They are about i or

PERTUSSIS

2/z long and tend to occur in pairs or short chains. Some claim that
they are Gram-positive.

Culturally the formation of little pits of liquefaction in Loffler's serum within
twenty-four hours which later become confluent may be regarded as fairly character-
istic. They do not grow on nutrient agar.

After two or three days on blood-serum rather marked involution
forms occur. While usually causing a more or less chronic conjuncti-
vitis they may at times produce a keratitis.

NOTE. — A Gram-negative bacillus which is less than i micron long, growing singly,
or in pairs, and known as the bacillus of Zur Nedden has been stated to produce
corneal ulcers. It grows readily on agar or other ordinary culture media. It
coagulates milk.

Bacillus of Chancroid (Ducrey, 1889). — These are short coccobacilli,
occurring chiefly in chains. They show bipolar staining. They grow
best in a mixture of blood and bouillon.

Material for culturing should be obtained before the lesion ulcerates. The
exudate should be inoculated on blood agar, i part blood to 2 of agar. After forty-
eight hours small glistening colonies develop which easily slide about the slant when
touched with a loop.

Bacillus of Bordet-Gengou. — This bacillus was reported as the cause
of whooping-cough by Bordet and Gengou in 1906. (Czaplewski and
Reyher had previously reported oval bipolar staining organisms, as the
cause of pertussis, and other authors influenza-like organisms.)

The bacillus is oval, Gram-negative, shows bipolar staining, somewhat resembles
B. influenza and grows only on uncoagulated serum media, as blood or ascites agar.
The original cultures are very scanty so that the colonies are difficult to recognize.
In subcultures the growth is more flourishing. The organism is only found in white,
thick, leukocyte abounding sputum, of the beginning of the disease. Hence per-
tussis is probably contagious only at the onset.

Complement binding and agglutination reactions have been obtained. For
diagnosis stain the sputum. Remember that pertussis gives a mononuclear leuko-
cytosis of 15,000 to 50,000.

For isolation from sputum the following medium is required. Auto-
clave 500 grams potato with 1000 c.c. of 4% glycerine solution. Pour
off excess of fluid. Emulsify potato in 1 500 c.c. normal salt solution and
add powdered agar to 3 or 4%. For use mix with an equal quantity
of defibrinated blood.

114 STUDY AND IDENTIFICATION OF BACTERIA

GRAM NEGATIVE BACILLI GROWING ON ORDINARY MEDIA

Bacillus Pneumonias (Friedlander, 1882). — This organism is re-
sponsible for about 5% of the cases of pneumonia. It is usually
termed the Pneumobacillus to distinguish it from the Pneumococcus;
at other times Friedlander's bacillus. The name of Fraenkel attaches
to the Pneumococcus. Morphologically, it is a short, thick bacillus,
and in pathological material, as sputum, shows a wide capsule. It is
nonmotile and Gram-negative. The colonies on agar are of a pearly
whiteness and are markedly viscid. On potato it shows a thick viscid
growth containing gas bubbles. The characteristic culture is the nail
culture of a gelatin stab. The growth at the surface is heaped up like a
round-headed nail, the line of puncture resembling the shaft of the nail.

It does not liquefy gelatin. It does not produce indol, and does not produce gas
in lactose bouillon — differences from the colon bacillus — with which it may be con-
fused in cultures, as it does not then possess a capsule. If in doubt, inject a mouse
at the root of the tail. Death from septicaemia occurs in two days. The peritoneum
is sticky and numerous capsulated bacilli are present in the blood and organs. The
organisms which have been isolated from rhinoscleroma and ozcena are practically
identical with the B. pneumonia. This group of organisms is generally referred
to as the Friedlander group. Similar organisms have been isolated from the dis-
charges of middle-ear diseases and in anginas. Cases have been reported where the
B. pneumonia was the cause of septicaemia in man.

Bacillus Pestis (Kitasato, Yersin, 1894).— This is the organism of
plague. It is primarily a disease of rats. It is the member of the group
of haemorrhagic septicaemias (Pasteurelloses), from which man suffers.

Other Pasteurelloses are chicken cholera, swine plague, mouse sep-
ticaemia and rabbit septicaemia. This is a widely distributed group and
may include saprophytic organisms as well as those noted for their
virulence.

B. cholera gallinarum and B. suisepticus are approximately similar in
size and cultural requirements to B. pestis. The oval bacillus with
bipolar staining in smears from tissues is very characteristic for both
of them. Another name for swine plague (B. suisepticus) is infectious
pneumonia of swine. The organism is chiefly found in the lungs. The
bacillus of plague was first isolated by Yersin from a plague bubo, in
1894, at Hong Kong. It is true that Kitasato reported a bacillus
which he had isolated from the blood of a plague patient, on July 7,
1894 (Yersin's report was made July 30, 1894). Kitasato's bacillus
was motile, Gram-positive, coagulated milk and gave a turbidity in

PLAGUE 115

bouillon, characteristics which were just the opposite of those of the
organism reported by Yersin.

Where the plague bacilli are found chiefly in the glands, we have bubonic plague;
when in lungs, pneumonic plague; when localized in the skin and subcutaneous tissue,
the cellulo-cutaneous; and when as a septicaemia, septicaemic plague. An intestinal
type is recognized by some authors. It must be remembered that in all forms of
plague the lymphatic glands show hemorrhagic oedema; it is in bubonic plague, how-
ever, that the areas of necrosis with periglandular oedema are prominent.

Where the symptoms are slight, mainly buboes, the term pestis minor
is sometimes used; the typical disease being termed pestis major. In
pneumonic plague we have a bronchopneumonia.

In smears from material from buboes, from sputum, or in blood smears, as well
as from blood or spleen smears from experimental animals, we obtain the typical

FIG. 3 1 .—Colonies of plague bacilli forty-eight hours old. (Kolle and Wassermann.')

morphology of a coccobacUlus (1.5X0.5*1) with very characteristic bipolar staining;
there being an intermediate, unstained area. Very characteristic also is the appear-
ance in these smears of degenerate types which stain feebly and show coccoid and
inflated oval forms. The presence of these involution forms associated with typical
bacilli is almost diagnostic for one with experience. Inoculating tubes of plain agar
and 3% salt agar with this same material, we obtain in plain agar cultures organisms
which are typically small, fairly slender rods, which do not stain characteristically
at each end and are not oval. The smear obtained from the salt agar presents most
remarkable involution forms— coccoid, root-shaped, sausage-shaped forms, ranging
from 3 to 12 microns in length, more resembling cultures of moulds than
bacteria. Another point is that on the inoculated plain agar we are in doubt at
the end of twenty-four hours whether the dewdrop-like colonies are really bacterial
colonies or only condensation particles. By the second day, however, these colo
have an opaque grayish appearance, so that now, instead of questioning the presi

n6

ST"UDY AND IDENTIFICATION OF BACTERIA

of a culture, we consider the possibility of contamination. Litmus milk is rendered
slightly acid but not sufficiently to change the lilac color. Glucose broth is made
slightly acid but there is no effect on lactose.

Blood cultures in septicsemic plague may show from 5 to 500,000 bacilli per c.c.
Smears from the blood in such cases are positive in only about 17%.

The plague bacillus grows well at room temperature — its optimum
temperature being 30° instead of 37°C., as is usual with pathogens.
Next to the salt agar culture, the most characteristic one is the stalac-
tite growth in bouillon containing oil drops on its surface. The culture
grows downward from the undersurface of the oil drops as a powdery

FIG. 32. — Pest bacilli from spleen of a rat. (Kolle and Wassermann.)

thread. These are very fragile, and as the slightest jar breaks them,
it is difficult to obtain this cultural characteristic.

While Klein states that B. coli, Proteus vulgaris and, in particular, B. bristolensis
may be mistaken for plague bacilli, if bipolar staining alone be relied upon, yet it is
B. pseudotuberculosis rodentium which may confuse an experienced worker. While
this latter is only moderately pathogenic for rats yet the fact that rats may be
immunized to B. pestis by inoculation with B. pseudotuberculosis rodentium brings
up the suspicion of identity of the two organisms. In diagnosing ^always use animal
experimentation. Owing to the difficulty in emulsifying plague bacilli, agglutination
tests are not satisfactory. B. tularense resembles B. pestis. See Part IV.

Albrecht and Ghon have shown that by smearing material upon the
intact, shaven skin of a guinea-pig, infection occurs. This is the crucial
test.

A pocket made by cutting the skin of a guinea-pig with scissors and extended
subcutaneously with scissors or forceps, into which a piece of the suspected plague

RATS AND PLAGUE H^

tissue is thrust with forceps, is more practical than injecting an emulsion with hypo-
dermic syringe.

Mice inoculated at the root of the tail quickly succumb. Rats, this being pri-
marily a disease of rats, are of course susceptible. Other rodents, as squirrels, are
susceptible. It has been suggested that a rodent, the Siberian marmot, or tarabagan
(Arctomys bobac} might be the starting-point of plague outbreaks. In natural
plague of rats, the lesions which establish a diagnosis even without the aid of a
microscope are dark red, subcutaneous injection of the flaps of the abdominal walls

FIG. 33. — Pest bacillus involution forms produced by growing on 3% salt agar.
(Kolle and Wassermann.)

as they are turned back, fluid in the pleural cavities, oedematous haemorrhagic
periglandular infiltration and swelling of the neck glands, and in particular a creamy,
mottled appearance of the liver.

The bacillus known as Danysz virus also causes whitish granules of
liver but these are larger and do not have the appearance as if peppered
on the liver.

The neck glands in rat plague are chiefly involved because the flea prefers to
inhabit the skin of the neck. The spleen is swollen, congested and granular and
smears from this viscus will show the bacilli.

A chronic rat plague, which may be a factor in keeping up the disease, is char-
acterized by enlargement of the spleen and the presence within it of nodules contain-
ing plague bacilli. McCoy has noted that the frequency of the cervical bubo in
rats, noted by the Indian Commission (72%), was not found in California. The
glands show periglandular infiltration and injection as well as enlargement.

Recent investigations in India have definitely determined the fact
that the flea (Xenopsylla cheopis) is the intermediary in the transmis-
sion of plague from rat to rat and from rat to man.

Il8 STUDY AND IDENTIFICATION OF BACTERIA

In Europe and U. S., Ceratophyttus fasciatus is the common rat flea and it, as well
as other species of flea, may transmit the disease. The bedbug will also transmit
plague. Fleas suck up the septicaemic blood of infected rats and there is a develop-
ment of plague bacilli in the cesophagus with more or less obstruction. When
feeding on man or other rats such fleas regurgitate and thus inoculate these plague
bacilli. The faeces of such fleas are also infectious.

In primary pneumonic plague the infective nature is very great and
appears to be by the respiratory atrium (from man to man). This
was the terrifying type of plague in the black death of the four-
teenth century.

Strong and Teague have shown that of 39 plates exposed before the mouths of
patients with pneumonic plague, with marked dyspnoea and pulmonary oedema, but
without coughing, only i plate showed plague bacilli. In 39 other experimental
plate cultures with coughing on the part of the patients there were 15 plates showing
plague bacilli.

The droplet method of infection is therefore the important one in plague pneu-
monia.

As these droplets are expelled to a considerable distance not only should the
respiratory inlets be protected by masks but the conjunctivas with glasses and abra-
sions with protective coatings.

For diagnosis make smears and cultures from material drawn from
a bubo by a syringe. (At a later stage, when softening begins, there
may not be any bacilli present.) Also, if pneumonic plague, from the
sputum. Blood cultures and even blood smears may be employed in
septicaemic plague. Formol fuchsin and Archibald's stain make satis-
factory stains. Always inoculate a guinea-pig with the material either
by rubbing it in with a glass spatula on the shaven skin or by sub-
cutaneous injection.

For prophylaxis the most important method is that of Haffkine. Stalactite bouil-
lon cultures of plague are grown for five to six weeks. These are killed by a tem-
perature of 65°C. for one hour. Lysol (M%) is added to the preparation and from
0.5 to 4 c.c. injected, according to the age and size of the individual treated. Sus-
ceptibility is reduced about one-fourth, and of those attacked after previous vaccina-
tion, the mortality is only about one-fourth of what it is among the noninoculated.
Strong prepares a prophylactic vaccine from living plague cultures rendered avirulent.
Yersin's serum, made by injecting horses with dead plague cultures and afterward
with living ones, is of value prophylactically and has possibly considerable curative
power.

The Eberth, Gartner and Escherich Groups. — From a standpoint
of cultures in litmus milk and sugar bouillon we can divide the organ-
isms related to typhoid at one extreme and the colon at the other into
three groups.

THE TYPHOID-COLON GROUP

119

1. The Eberth or typhoid group. There are three important patho-
gens in this group: the B. typhosus, the B. dysenteric, and the B. facalis
alkaligenes. The color of litmus milk is practically unaltered and there
is no gas production in either glucose or lactose bouillon. No coagu-
lation of milk. No reduction of neutral red. The B. typhosus and
the B. facalis alkaligenes are actively motile, while the B. dysenteries
is nonmotile or practically so.

During the first twenty-four to forty-eight hours there is a moderate acid produc-
tion by typhoid, so that the milk culture is less blue, while with the B. facalis alka_
ligenes the alkalinity is intensified from the start, so that the blue color is deepened^

2. The Gartner or hog cholera group. Besides organisms important
for animals and probably at times for man, such as B. cholera suum
and B. psittacosis and B. icteroides (interesting historically as having
been reported as the cause of yellow fever by Sanarelli), we have two
pathogens: i. B. enteritidis (Gartner's bacillus) and 2. B. paratyphoid
B. In this connection it may be stated that the present view is that
hog cholera is caused by an ultra-microscopic organism and not by the
B. cholera suum.

These organisms cannot be separated culturally, but only by immunity reactions.
They do not turn litmus milk pink. They produce gas in glucose bouillon, but not
in lactose. They very powerfully reduce neutral red with the production of a yellow-
ish fluorescence. They do not coagulate milk. There is a transient acidity in the
litmus milk, but becoming shortly afterward alkaline, the lilac-blue color is intensified.
Both organisms are motile.

3. The Escherich or colon group. These turn litmus milk pink,
coagulate milk, reduce neutral red, and show varying degrees of motil-
ity. The three groups of organisms just described are nonliquefiers of
gelatin. Two intestinal organisms, the B. cloaca and the Proteus vul-
garis, differ in liquefying gelatin.

Bacillus Typhosus (Eberth, 1880; Gaffky, 1884). — This organism
may be isolated from the stools, urine, and the blood of typhoid patients.

At postmortem it can be best isolated from the spleen, but is also present in
Peyer's patches which have not ulcerated. When ulceration has occurred contami-
nation with B. coli, is almost sure. Cultures may be obtained from the liver also.
In sections made from spleen the Gram-negative bacilli are apt to be decolorized.
Thionin, then blotting and clearing in oil or xylol, shows the clumps of bacilli lying
between the cells.

Formerly it was supposed that by the differences in the thickness of the film of
a colony or by its varying shades of grayish-blue, we possessed data of importance in
differentiating typhoid from related organisms.

120 STUDY AND IDENTIFICATION OF BACTERIA

The colonies look like grapevine leaves.

Growth on potato was also considered as affording information. At present,
the biochemical reactions give us information assisting in differentiation, and the
agglutination and bacteriolytic phenomena, the final diagnosis. The various plating
media are considered under media for plating out faeces.

Not only do we find hyperplasia of the endothelial cells in the lymphoid tissue
of Peyer's patches and the mesenteric glands and the spleen, with subsequent necro-
ses, but focal necroses of the same character are found in the liver.

A striking feature of the pathology of typhoid fever is the long-con-
tinued persistence of the organisms in the gall-bladder and elsewhere.
It is beginning to be believed that a previous typhoid infection, pos-
sibly so mild as to have passed unnoticed, is at the basis of gall-bladder
infections and resulting gall-stones. Various bone infections, especially

FIG. 34. — Seventy-two-hour-old culture of typhoid bacillus on gelatin. (Kolle and

Wassermann.)

osteomyelitis, have shown the typhoid bacilli in pure culture. For-
merly it was supposed that the typhoid bacillus brought about its lesions
by a local infection centered in the ileum. The present view is that
typhoid bacilli effect an entrance into the blood stream through some
lymphoid channel, as by tonsil or other alimentary lymphoid structure.
Of animals, only the chimpanzee seems to be susceptible.

They develop in the general lymphatic system, the spleen in partic-
ular, where they are protected from the bactericidal power of the blood.
After a time, however, approximately the period of incubation, they
become so abundant in these lymphatic organs that they are carried
over into the general circulation. Then as a result of bacteriolysis the
intracellular toxins are liberated and symptoms develop. If bacteri-

TYPHOID BACILLI IN THE BLOOD

121

olysis takes place other than in the blood we have various suppurative
processes. As a result of the formation of antibodies, the development
in spleen, etc., is checked but should. these immunity reactions become
less potent relapses may occur or various local infections manifest
themselves.

As the bacilli do not multiply to any extent in the blood itself the disease cannot
be considered as a typical septicaemia but as a bacterisemia.

Animals are not susceptible to typhoid fever with the possible exception of the
higher apes. Of course the injection of living or dead cultures may kill an animal
but there are no characteristic localizing symptoms.

FIG. *<;.— Bacillus of typhoid fever, stained by Loffler's method to show flagella.

(Xiooo.) (Williams.}

Typhoid bacilli can be isolated from the blood during the latter
period of incubation and rarely after the tenth day of the disease. It
is a practical point that the time to isolate the bacteria from the blood
is in the first days of the attack. The diagnosis by agglutination is
only expected after the seventh to tenth day. Agglutination may not
appear until during convalescence, and in about 5% of the cases it is
absent. It, as a rule, disappears within a year.

Very little success has been obtained with curative sera. Chantamesse, by
treating horses with a nitrate from cultures of typhoid bacilli on splenic pulp and
human defibrinated blood, claimed to have obtained a curative serum possessing

122 STUDY AND IDENTIFICATION OF BACTERIA

antitoxic power. Wright's method of prophylactic inoculation is now being em-
ployed in the British army with apparent success. In this, twenty-four- to forty-
eight-hour-old cultures are killed at 53°C.; K% °f lysol is then added. An injec-
tion of 500,000,000 bacteria is made at the first inoculation, and ten days later an
injection of 1,000,000,000. The British prefer to inject subcutaneously in the in-
fraclavicular region and at the insertion of the deltoid. The Germans consider 3
injections as conferring greater immunity.

Russell has obtained splendid results in the U. S. Army with his method of
vaccination. In this 3 injections are given at intervals of ten days, the dosage
being 500,000,000, for the first and 1,000,000,000 for each of the 2 succeeding
injections.

Typhoid vaccines sterilized with 0.5% of phenol appear to keep much longer and
to have a higher immunizing power than those prepared by sterilization with heat
and subsequent addition of the antiseptic.

Typhoid bacilli may be found not only in the blood, urine and faeces but as well
in the sputum of cases showing pulmonary involvement. They have also been found
in the cerebrospinal fluid of cases showing meningeal symptoms. At the autopsy
they may be found in the spleen, Pyer's patches, mesenteric glands and liver.

A very important discovery is that certain persons, who may have had only a
slight febrile attack, may eliminate typhoid bacilli for years in their faeces (typhoid
carriers) . The bacilli are also eliminated for considerable periods in the urine. Dis-
tinction is now being made between acute carriers (convalescents) and chronic
carriers.

In experiments on higher apes there was evidence that the bacilli eliminated by
carriers are in many instances nonpathogenic. About one-half of typhoid cases are
believed to be due to contact infections. Drigalski gives it for Germany as 64.7%.
The water transmission factor is of less importance than was formerly stated.

The most satisfactory method of detecting carriers is by examination
of faeces or urine plated out on Endo's medium. While carriers usually
give a Widal reaction this is by no means constant. Typhoid carriers
are said to maintain a high opsonic index.

The urine and faeces of typhoid convalescents should be proven negative by cul-
tural procedure before discharging the patients.

Vaccination may possibly be a satisfactory measure in bringing about the dis-
appearance of typhoid bacilli in the dejecta of carriers.

For laboratory diagnosis, blood cultures during the first week and
agglutination tests during the second week and onward are the practical
methods.

Along with the agglutination tests the urine and faeces should be cultured on
Endo's plating medium and later transferred to Russell's medium for cultural
identification. The positive identification, provided the culture so isolated shows the
cultural characteristics of typhoid, is made by testing the bacilli for agglutination
with a known typhoid serum. Instead of the usual blood cultures one may use the

PARATYPHOID 123

clot in the Wright U-tube for culturing and the serum remaining after centrifugaliza-
tion for the Widal test (clot culture). B. typhosus appears in the blood in relapses.
Kayser considered that about 27% of cases of typhoid in Strasburg were caused by
raw milk, 17% by contaminated water, 17% by contact with typhoid, and 10% were
due to typhoid carriers. Other cases were due to infected food, and about 13% were
of origin impossible to determine. These latter may have been due to unrecognized
typhoid carriers. He does not attach the same importance to fly dissemination as
do American authors.

Contact infection is the great factor in perpetuating typhoid fever
but this agency shows diminishing cases each year provided water and
milk supplies are safe. The leading European cities as a result of a
safe water supply rarely show more than about 3 typhoid deaths per
100,000 population per year. Edinburgh shows less than one per
200,000 for the year 1910. In American cities rates of 12 to 15 per
100,000 are common.

The Gartner or Meat-poisoning Group. — Under this designation
may be considered the organisms which cause gastrointestinal disorders
of varying degrees, infection with which is usually brought about by the
ingestion of meat obtained from diseased cattle. Unless the meat is
thoroughly cooked the bacilli in the interior may not be killed.

In this group may be placed B. enteritidis, the typical meat-poisoning organism,
B. paratyphoid B, B. Danysz, B. Aertryck, B. typhi murium and B. suipestifer.

B. suipestifer or the hog cholera bacillus was formally thought to be the cause of
this important epizootic. It is found in the intestines of quite a percentage of
healthy hogs. The cause is now known to be a filterable virus.

These organisms are alike morphologically and culturally and show quite a
tendency to bipolar staining and reduction of neutral red with fluorescence in
forty-eight hours. B. paratyphoid B, B. Aertryck and B. suipestifer are alike from
an agglutination standpoint, while B. enteritidis and B. Danysz show similarity
in this respect. B. paratyphoid A stands by itself.

Paratyphoid Bacilli (Achard and Bensaude, 1896; Schottmiiller,
1901). — Cases resembling mild attacks of typhoid occasionally show
agglutination for paratyphoid bacilli. These organisms have also been
isolated from the blood, as with typhoid. Two types have been recog-
nized: the paratyphoid A. and the paratyphoid B. The latter occurs
in 80% of such cases. Culturally, paratyphoid B. cannot be separated
from Gartner's bacillus. In paratyphoid A. there is less gas produced
in glucose bouillon than with paratyphoid B., and the primary acidity
of litmus milk is not succeeded by a subsequent alkalinity. It does
not seem practical to draw a fine distinction between these two strains.

124 STUDY AND IDENTIFICATION OF BACTERIA

Paratyphoid B. not only gives symptoms resembling a mild typhoid infection,
but may show symptoms more like those of meat poisoning or even cholerine. It
is more pathogenic for laboratory animals than is B. typhosus. The development of
antibodies upon immunizing a man or animal with paratyphoid organisms does not
seem to approach that obtained with typhoid.

Castellani has conducted experiments with typhoid and paratyphoid vaccines
and has found that typhoid vaccines give an agglutinating serum of about i to 350
titre from the second to fifth week dropping to about i to 100 after three or four
months. Paratyphoid A. gives one of about i to 75 for the first month which drops
to about i to 60 after four months. Paratyphoid B. gives about one-half the
agglutination response that paratyphoid A. does.

Bacillus Enteritidis (Gartner, 1888). — This organism has been fre-
quently isolated from cases of gastroenteritis from ingestion of infected
meat.

Meat from healthy animals which has been in contact with that of diseased
animals may become infected. The simple act of placing a piece of infected meat
on a sound piece may infect the latter. It has been noted that the bacteria, or their
toxins, may be distributed unevenly in the meat eaten, so that one person consuming
the same meat may be made very ill while others eating this meat may escape
infection. Infection of food may occur not only from unclean handling but from the
material carried by flies or even from the faeces of mice or rats deposited on
foodstuffs.

This organism is very pathogenic for laboratory animals, producing a haemor-
rhagic enteritis and at times a septicaemia. Where meat has been contaminated with
Gartner's bacillus toxins may have been produced, and symptoms of poisoning with
acute gastroenteritis would occur shortly after ingestion. This is not a true toxin
as it does not require a period of incubation before manifesting its toxic action. It
is interesting to note that this toxin is not destroyed by the boiling temperature, thus
differing from the toxin of the other important meat-poisoning (botulism) bacillus —
B. botulinus — which is rendered innocuous by a temperature of 65° or 7o°C. If
there is only a little toxin introduced with the contaminated meat, the symptoms
will be delayed one or two days. Such organisms have been isolated in pure culture
from cases with high fever, marked intestinal derangement, with considerable blood
in the rather fluid stools. In two cases studied the disease was at first diagnosed
as a severe typhoid infection. Klein thinks the organism of Danysz's virus (to
kill rats during plague epidemics) may be identical with B. enter itidis.

Proteus Vulgaris. — This organism is often encountered in plates
made from faeces, or sewage contaminated water.

It is common in decaying meat or cheese, and cases of even fatal poisoning
with marked gastrointestinal symptoms and cardiac failure have been reported.
At times it is the cause of cystitis. The colonies on agar are moist and unevenly
spreading (amoeboid). The bacilli are very motile, long and slender, tend to form
filaments and, as a rule, are Gram-negative. It digests blood-serum and is a rapid
liquefier of gelatin. In litmus milk it coagulates with a soft clot and an alkaline

DYSENTERY

125

reaction. Subsequently the litmus is reduced and the clot digested giving a dirty
yellowish-brown fluid. Indol is rarely produced. The cultures generally have a
putrefactive odor. In infective jaundice (Weil's disease) this organism has been
reported as the cause. Organisms of this group were formerly designated as B.
termo.

Bacillus Dysenterise (Shiga, 1898). — Dysentery bacilli produce a
coagulation necrosis of the mucous membrane of the large intestine
and occasionally of the lower part of of the ileum. Polymorphonu-
clears are contained in the fibrin exudate.

It was formerly thought that these lesions were of local origin, but the present
view is that toxins are produced which, being absorbed, are eliminated by the
large intestine with resulting necrosis. Flexner, by injecting rabbits intravenously
with a toxic autolysate, produced characteristic intestinal lesions. The toxin with-
stands a temperature of 7o°C. without being destroyed. The toxin may cause
joint trouble.

There are two main types of dysentery bacilli:

1. Those producing acid in mannite media — the acid strains (Flex-
ner-Strong types).

2. Those not developing acid in mannite (Shiga-Kruse types).
Ohno finds that fermentative reactions do not correspond to immunity
ones. Thus an acid strain used to immunize a horse may produce a
serum more specific for a nonacid strain. Hiss, however, found that
organisms similar in fermentation reactions agreed in agglutination
ones. The Shiga type is very toxic in cultures, while the Flexner type
does not seem to possess a soluble toxin.

Clinically the toxaemia of cases of dysentery due to Shiga types is marked while
that from Flexner strains is slight.

We often designate the Shiga strains as the toxic group and the acid-producing
strains as the nontoxic. The "Y" type of organisms also produces acid in mannite.
The following is the Lentz table:

Lentz recognizes 4 types of dysentery bacilli for the differentiation of which he
uses mannite, maltose and saccharose bouillon with litmus as an indicator.

Shiga-Kruse

Flexner

Strong

« Y"

Mannite

Blue

Red

Red

Red

Maltose

Blue

Red

Blue

Blue

Saccharose

Blue

Blue

Red

Blue

A strain which ferments not only mannite, dextrose, maltose and saccharose,
but dextrin as well, is known as the Harris type.

126 STUDY AND IDENTIFICATION OF BACTERIA

The Shiga strains are apt to cause a paresis of the hind extremities of the injected
rabbit which may be followed by^ paralysis and death. At the Lister Institute
injections of a soluble toxin produced a serum of marked antitoxic power. Such a
dysentery serum, which is probably both antitoxic and antimicrobic, is of curative
value. Shiga immunized horses with polyvalent cultures and obtained a polyvalent
serum which has reduced the death rate about one-third.

The dysentery bacillus is present in the milky white, leukocyte filled
blood flecked mucous stools during the first five or six days of the dis-
ease. By the tenth day it has probably disappeared. Lactose litmus
agar is the most satisfactory plating medium. The stool of the first
two days may give practically a pure culture. The staining of a smear
from the muco-purulent stool is rich in phagocytic cells, many of them
packed with Gram-negative bacilli. In all cultural respects the dysen-
tery bacillus resembles the typhoid, and the only practical method of
distinguishing these two organisms, other than by agglutination reac-
tions, is by the nonmotility or exceedingly slight motility of the dysen-
tery bacillus.

The characteristic of nonmotility is of greatest differentiating value and the
reports of slight motility are probably from misinterpretation of molecular movement
as motility. The dysentery bacilli do not form those threads or whip-like filaments
so characteristic of typhoid cultures and are somewhat plumper.

As a rule they occur singly or in pairs, and of rather oval or even coc-
coid shape and may stain bipolarly. The colonies are much like those
of typhoid. Type "Y" colonies often show indentation while the
Shiga types show round colonies.

The dysentery bacillus is not found in the blood and hence is not eliminated in
the urine. It is found in mesenteric glands. In dysentery patients agglutination
phenomena do not show themselves until about the twelfth day from the onset.
Hence, this procedure is of no particular value in diagnosis. It is of value, however,
to identify an organism isolated from the stools at the commencement of the at-
tack, using serum from an immunized animal or a human convalescent for the
agglutination test.

Butler has suggested taking serum from dysentery convalescents, noting the
strain involved, and preserving it by taking up with filter-paper as recommended
by Noguchi for the Wassermann haemolytic amboceptor. This I consider very
valuable as it is very difficult to immunize rabbits with a Shiga strain on account
of its great toxicity. Dean has reduced the toxicity of Shiga vaccines by treat-
ing with eusol.

There seems to be very little agglutination power in the serum of convalescents
from Shiga strains. Flexner strains give agglutination, but early in convalescence
the serum is not apt to have a titre of more than i to 50.

COLON BACILLUS

127

Morgan has reported as the cause of certain cases of bacillary dysen-
tery a bacillus known as B. Morgan, No. i. It is motile, produce indol,
and in glucose bouillon gives a very slight amount of gas.

It does not change mannite and does not produce a primary acidity in litmus
milk. This organism is a frequent cause in England of summer diarrhoea of
children. Flies from houses with such cases often show Morgan's bacillus. A
dysentery type much like the Flexner-Strong strain is often found in the enteric
affections of children in the United States.

In Japan, dysentery-like epidemics of a very fatal disease, termed
ekiri, occur among young children. The organism is very motile, pro-
ducing gas and acid in glucose but not in lactose media. It is reported
at times to show indol production. Apparently a member of the Gart-
ner group.

More recently a strain of dysentery bacilli, known as Type Y, has been con-
sidered of importance. This organism is very closely related to the Flexner strain
and only differs from it in that it requires about forty-eight hours to turn mannite
litmus media pink and that maltose litmus remains blue. An organism showing
similar cultural characteristics has been recently recovered from faeces of laboratory
rabbits by German workers investigating the problem of whether certain animals
might serve as carriers for dysentery.

B. COLI, B. LACTIS AEROGENES, B. CLOACA

While the COLON BACILLUS chiefly inhabits the large intestine, the B.
lactis aerogenes is to be found in the upper part of the small intestine.
While they may be separated on the ground of motility, yet it is by the
greater fermentative activity of the B. lactis aerogenes that they are
best separated. Some consider them as only representing different
strains of the same organism.

The main points of distinction are gas production in starch media (gas bubbles
on potato slant) and frequent nonproduction of indol. B. lactis aerogenes is closely
related to the pneumobacillus and at times shows capsules. It is best differentiated
from the pneumobacillus by its gas production in lactose and coagulation of milk.

Some consider that the B. coli produces a bactericidal substance which
inhibits the growth of, or destroys, pathogenic bacteria which may have
passed the destructive influences of the gastric juice; others that this
effect is due to their free growth and the development of phenol and
various putrefactive substances.

The probable importance of the colon bacillus in protecting the ^organism is
shown by the fact that where numerous colonies of pathogenic organisms may be

128 STUDY AND IDENTIFICATION OF BACTERIA

cultivated from faeces we may find a diminution in number or absence of the colon
bacillus. This condition may be observed in infections with the organisms of
dysentery, cholera, typhoid, and paratyphoid. While its normal function is prob-
ably protective, yet the B. coli is an important pathogenic agent, it being fre-
quently the organism isolated from purulent conditions within the abdominal cavity,
especially in appendicitis and lesions about the bile ducts. It is particularly prone
to cause lesions of the bladder and pelvis of the kidney. In the treatment of colon
cystitis by vaccines of dead colon bacilli, brilliant results in opsonic therapy have
been obtained.

Sir A. Wright thinks that certain cases of mucous colitis may be due to colon
infection and that vaccination may cure them. The colon bacillus is fully con-
sidered under the bacteriology of water.

B. cloaca was isolated first from sewage by Jordan. It is, as a rule,
a rapid liquefier of gelatin, and in its reactions with sugars and litmus
milk resembles the colon bacillus.

Where the gelatin liquefaction is slow or slight B. cloaca may be dis-
tinguished from B. coli by its gas formula which is about three times as
much CO2 as H, just the reverse of that of the colon bacillus. B. lactis
aerogenes is also often found in sewage. It is one of the causes of the
souring of milk.

B. ACIDOPHILUS, B. BlFIDUS, B. BULGARICUS

These are often termed the long rod group of lactic acid bacteria in
contradistinction to certain other Gram-positive bacilli which are short
and oval and which are confused with the so-called milk streptococci.

The long rod group often forms chains and often shows metachromatic granules
which stain with Neisser's method. They are readily distinguished from Gram-
negative lactic acid producers, of which the type is B. lactis aerogenes, by their
Gram-positive staining. B. acidophilus often give the impression of a diphtheroid
in a Gram stained faeces smear. It is nonmotile and often shows polar granules.
Grows only at temperatures above 22°C., op. 37°C. It grows better anaerobically
than aerobically and then shows the clubbed involution characteristics of B. bijidus;
so that some consider these organisms the same, the morphology of B. bijidus being
the result of anaerobiosis. Original cultures are best made in i% glucose and i%
acetic acid bouillon. Some authorities consider B. bijidus the most important
representative of the large intestine flora. B. lactis acidi is less thermophilic than
B. acidophilus and coagulates milk which B. acidophilus does not do. Certain
polar granule bacteria, as B. granulosum, found in Yoghurt, are similar to B. acido-
philus but coagulate milk; no gas. B. bulgaricus is the type of the group and is
discussed under milk.

Rodella thinks B. acidophilus, B. bijidus, B. gastrophilus and the Boas-Oppler
bacillus identical. B. bulgaricus is said to never show polar granules. B. bulgaricus
and the group of organisms similar to it found in buttermilk, etc., are widely used in

CHROMOGENS I2Q

the treatment of various intestinal troubles. North has used cultures of B bulgaricus
for extermination of undesirable organisms in other parts of the body than the
alimentary canal (used as applications in nasal, throat or genito-urinary infections).

CHROMOGENIC BACILLI

These are identified by the color of their colonies on agar. The
B. pyocyaneus is the most important one of them in medicine, but the
B. prodigiosus is also of interest medically. A violet chromogen, the
B. yiolaceus, which is motile and liquefies gelatin, has been described
under many names. It has been found in water.

An orange-yellow chromogen, the B. fulvus, is nonmotile and varies as to its
liquefaction of gelatin.

B. pyocyaneus (Gessard, 1882).— This organism is frequently termed
the bacillus of green or blue pus. It is a small (2. 5X0.5/4) motile
Gram-negative bacillus.

It is generally a slender delicate bacillus often showing thread-like
arrangement but at times it may appear as short plump rods. It grows
readily at room or incubator temperature. It liquefies gelatin rapidly.
The green color diffuses through the agar or gelatin on which it grows,
so that we not only have the green-colored colony, but the medium as
well is colored. Upon potato the colonies are more of a deep olive
green to dirty brown.

No gas is produced in either glucose or lactose bouillon; blood-serum is digested,
the pitted surface showing a reddish-brown color. The protein ferment pyocyanase
has been used to remove diphtheritic membrane and for treatment of M, catarrhalis
nasal catarrhs. There are 2 pigments — a green water soluble one and a blue
one soluble in chloroform.

It is widely distributed in water and air, and is frequently isolated
from faeces. The B. fluorescens liquefaciens of water seems to be simply
a strain of B. pyocyaneus. The B. pyocyaneus is frequently associated
with other pus organisms in abdominal abscesses.

In addition to having an endotoxin, it produces a soluble toxin similar to diph-
theria toxin. This toxin differs from those of diphtheria and tetanus in that it can
stand a temperature of ioo°C., while those of diphtheria and tetanus are destroyed
at about 65°C. The fact that the union between toxin and antitoxin is only of a
binding, neutralizing nature is best shown by taking a mixture of pyocyaneus toxin
and antitoxin which is innocuous and heating it. This destroys the antitoxin, but
does not injure the toxin. We now find that the original toxicity has returned. The
antitoxins of diphtheria and tetanus are more stable than the corresponding toxins;
9

130 STUDY AND IDENTIFICATION OF BACTERIA

hence, this experiment would be impossible with them, as upon heating we should
first destroy the toxin.

On account of the frequent association of B. pyocyaneus with other
organisms of better recognized pathogenicity it has until more recently
been considered rather harmless; this view can no longer be entertained
as it is frequently the sole cause of middle-ear inflammations, intestinal
disorders, cystitis and possibly at times of septicaemia.

B. Prodigiosus. — This is a very small coccobacillus which shows mo-
tility in young bouillon cultures. It is Gram-negative. The colonies
on agar or other solid media show a rich red color. The pigment only

FIG. 36. — Bacillus pyocyaneus. (Kolle and Wassermann.)

develops at room temperature, it is absent in cultures taken out of the
incubator. The B. prodigiosus is frequently found on foodstuffs, espe-
cially bread, where it may simulate blood. It liquefies gelatin rapidly
and gives a diffuse turbidity to bouillon. It is probable that B. indicus
and B. kilensis are strains of B. prodigiosus.

Coley's fluid, which has been used in cases of inoperable sarcoma and other
malignant growths, is a culture prepared by growing very virulent streptococci in
bouillon for ten days. This Streptococcus culture is now inoculated with B. prodi-
giosus, and after another ten days the mixed culture is killed by heat at 60° C. and
the sterile product injected. Coley injected about J^o c.c. of this vaccine. At
present he uses nonfiltered, heat sterilized bouillon cultures of a Streptococcus
obtained either from a case of erysipelas or septicaemia. To this is added material
from agar cultures of B. prodigiosus, grown separately and sterilized before adding
to the sterilized streptococcus bouillon culture.

CHAPTER IX

STUDY AND IDENTIFICATION OF BACTERIA. SPIRILLA.
KEY AND NOTES

KEY to recognition of gelatin liquefying, motile and Gram-negative
spiral or comma-shaped organisms.

A. Do not give the nitroso-indol reaction with sulphuric acid alone in twenty-four
hours.

1. Produce an abundant moist cream-colored growth on potato at room tempera-
ture.

(a) Finkler and Prior's spirillum (Vibrio proteus). Liquefaction of gelatin
very rapid. No air-bubble appearance at top of liquefied area. Cultures
have foul odor. Milk coagulated. Thicker spirillum than cholera. Iso-
lated from cholera nostras.

2. Scanty growth or none at all on potato at room temperature. Only a mod-
erate yellowish growth when incubated about incubator temperature.

(a) Spirillum tyrogenum (Deneke's spirillum). Does not liquefy gelatin so
rapidly as Finkler Prior. Thinner and smaller spirillum than cholera.

B. Give the nitroso-indol reaction with sulphuric acid within twenty-four hours.

1. Very pathogenic for pigeons.

(a) Spirillum metschnikovi. Liquefies gelatin about twice as rapidly as
cholera. Gives bubble appearance at top of stab.

2. Scarcely pathogenic for pigeons.
(a) Spirillum cholerae asiaticae.

Nonmotile, nonliquefying and Gram-positive spirilla have also been described.
There is also a large group of phosphorescent spirilla.

Spirillum Cholerae Asiaticae (Koch, 1884). — Typically, the morphol-
ogy of this organism is that of the comma (Comma bacillus of Koch).
It also frequently shows S shapes, and often appears in long threads
showing turns. When freshly isolated from cholera material they, as
a rule, show a fairly typical morphology, but after subcultures in the
laboratory variations are common, so that rod forms and round in-
volution shapes give a picture altogether at variance with the comma
shape.

Even in recent cultures of undoubted cholera we may have different types, as
coccoid forms and slender rods. Ohno has noted the fact that the same strain of
cholera will give at one time vibrio forms and again coccoid or rod forms, depending

132

STUDY AND IDENTIFICATION OF BACTERIA

on the reaction of the media. Inasmuch as the recognition of vibrio shapes is of
importance in diagnosis he recommends that material from a stool be inoculated
into 3 tubes of peptone solution of reaction +0.3, —0.5 and — i, respectively, one
of which would probably show vibrio morphology.

FIG. 37. — Cholera spirilla. (Kolle and Wassermann.)

The cholera spirillum is very motile (a scintillating motility) and
liquefies gelatin fairly rapidly, although more slowly than any of the
spirilla mentioned in the key.

*~v

FIG. 38. — Involution forms of the spirillum of cholera. (Van Ermengen.)

The colony on gelatin was formerly considered characteristic, but like most
cultural characteristics, it is now considered as being only of confirmatory value; it
is not specific. These colonies show in twenty-four hours as small granular white
spots which have a spinose periphery. An encircling ring of liquefaction now
makes its appearance and the highly refractile (as if fragments of sparkling glass)
colony can be separated into a granular center, a striated periphery, and a clear
external ring of liquefaction.

CHOLERA

133

On gelatin stabs the liquefaction produces a turnip-like hollow at the top of the
puncture — the air bubble appearance. It gives the nitroso-indol reaction with sul-
phuric acid alone (cholera red). Kraus attaches importance to the fact that cholera
does not produce a haemolytic ring on blood agar as do the pseudocholera spirilla-
a difficulty is that many pseudospirilla do not haemolize. Furthermore, true cholera
strains may occasionally show haemolysis, especially in laboratory cultures. Quite
a discussion has arisen in connection with a spirillum isolated from cases of diarrhoea
(no symptoms of cholera) in pilgrims at El Tor. This organism gave the immunity
reactions (agglutination) of true cholera but on account of its haemolytic power has
been considered as distinct from cholera. Such a view would seem to be untenable.
Sp. cholera grows very rapidly on peptone solution and this is
the medium for the enrichment test to be later described. On
this it may form a pellicle. On agar the colony is more opales-
cent (more of a translucent grayish blue) than the typhoid. It
does not grow on potato except at incubator temperature. It
does not coagulate or turn acid litmus milk. Some strains,
however, do produce a certain amount of acid. Using the Hiss
serum sugar media our strains produced acid in glucose and
saccharose but not in lactose. No gas production in any of the
sugars. The spirilla are found in myriads in the rice-water
discharges, these white flakes being desquamated epithelial
cells. They penetrate the crypts of Lieberkuhn, but rarely ex-
tend to the submucosa. The symptoms are due to an endo-
toxin.

Cholera may be transmitted from water supplies,
when the outbreak is apt to be widespread and in
great numbers from the start. Also by indirect con- FlG 39._spi-
tagion, as by flies or on lettuce, etc. A very im- rillum of cholera
portant point is that we have well persons whose g^atin^two^day"

fseces contain virulent cholera spirilla (cholera old. (Fraenkel

. and Pfeiffer.)

carriers).

Cholera spirilla disappear from the stools of cholera patients very
rapidly, usually in five to ten days.

Only exceptionally are organisms excreted longer than three or four weeks, but cases
are on record of periods approximating two or three months. Cholera carriers in good
health may come down with cholera as the result of administration of purgatives
or alimentary canal disorder. This would explain periods of incubation longer
than the usual one of two to five days.

Cholera carriers are therefore of less importance epidemiologically
than typhoid carriers, where carrier stage may last years.

It is well to remember, however, that cases have been reported of positive findings
after a period approximating two months from the onset of the attack of cholera.
Another important consideration is that the vibrios may be absent at one examina-
tion and be present at a later one. Purgatives seem to influence the reappearance

134 STUDY AND IDENTIFICATION OF BACTERIA

of the spirilla. An acid reaction of the faeces, such as that induced by lactic acid
bacteria, would apparently be of value in the prophylaxis of cholera carriers.

Greig has found infection of the bile of the gall-bladder or ducts in 80 cases in
271 cholera autopsies. While cholera spirilla are soon crowded out by intestinal
bacteria, thus explaining the short period during which cholera spirilla are excreted
by convalescents, this is not true when the cholera vibrio gets into the bile ducts or
gall-bladder, where ideal conditions prevail for a prolonged life. In fact bile has
recently been recommended as a selective medium for cholera enrichment. Greig
found one cholera convalescent excreting cholera vibrios forty-four days after the
attack. Of 27 persons who had been in contact with cholera patients 6 were ex-
creting cholera vibrios though apparently well.

To identify such spirilla immunity reactions are necessary:

1. Injected intraperitoneally into guinea-pigs, it produces a peritonitis and sub-
normal temperature. This reaction exists for spirilla other than the true cholera
spirillum.

2. Intramuscular injections into pigeons are only slightly pathogenic, if at all.

3. The agglutination test is the most practical. In this we use serum from an
immunized animal, in dilution of from 100 to 1000. It is rare that true cholera
vibrios fail to agglutinate in serum of i to 500 and even sera of i to 10,000 dilution
give the reaction. Serum of cholera convalescents may show agglutination as early
as the tenth day; it is usually best shown about the third week. Dunbar's quick
method is very practical. Make two hanging-drop preparations, using mucus from
the stool as the bacillary emulsion. To one add an equal amount of a i : 50 normal
serum; to the other a i : 500 dilution of immune serum. Cholera spirilla remain
motile in the control, but lose motility and become agglutinated in the preparation
with the immune serum.

4. Pfeiffer's phenomenon. If cholera spirilla are introduced into the peritoneal
cavity of immunized guinea-pigs (or if together with a i : 1000 dilution of immune
serum the mixture is injected intraperitoneally into normal guinea-pigs) and at
periods of ten to sixty minutes after injection, material is removed by a pipette from
the peritoneal cavity, the spirilla have lost motility, have become granular and
degenerated. Pseudospirilla are unchanged. This reaction may be carried on in
a pipette, using fresh serum.

Antisera for the treatment of cholera have not proved successful.

On the whole the reports from the use of anticholera sera are not
very encouraging. Savas, however, was favorably impressed by such
treatment during the Balkan war. It should be administered intra-
venously and early in the attack and given in doses of 50 c.c. Of 61
severe cases so treated the mortality was 55.7%. Of 17 severe cases
not receiving serum treatment all died.

Prophylactically, there are three prominent methods: i. That of Haffkine, where
live cholera spirilla are injected subcutaneously; and 2. Strong's cholera autolysate.
In this cholera cultures are killed at 6o°C. The killed culture is then allowed to
digest itself in the incubator at 37°C. for three or four days (peptonization). The
preparation is then filtered and from 2 to 5 c.c. of the filtrate is injected. Ferran
was the first to use vaccines.

DIAGNOSIS OF CHOLERA 135

3. At present the same methods are being used for cholera vaccines as for those of
typhoid. An emulsion of the vibrios in salt solution or a bouillon culture is subjected
to a temperature of S4°C. for one hour. Three doses are injected seven to ten days
apart, going from 500,000,000 to 2,000,000,000.

Among the Greek forces 0.45% of the inoculated and 1.9% of the noninoculated
were attacked. The inoculated, however, were sanitary troops and hence more
exposed to infection.

For diagnosis: i. take a fleck of mucus, make a straight smear and
fix; stain. with a i :io carbol fuchsin. The comma-shaped organisms
appear as fish swimming in a stream.

2. Inoculate a tube of peptone solution. The cholera spirilla grow
so rapidly, and being strong aerobes, they grow on the surface of the
fluid so that by taking a loopful from the surface, we may in three to
eight hours obtain a pure culture. Should there be a pellicle present,
this should be avoided in the transfer by tilting the tube slightly, so that
the material near the surface be obtained without touching the pellicle.
Inoculate a second tube from the surface of this first and, if necessary,
a third (enrichment method).

3. Test for cholera red reaction. (Simply adding from 3 to 5
drops of concentrated chemically pure sulphuric acid to the first or
second peptone culture after eighteen to twenty-four hours' growth.
Some specimens of peptone do not give the reaction.) At times we
only get the cholera red when we have a pure culture of cholera.

4. Smear a fleck of mucus or, better, the three-hour surface growth of
a peptone culture on a dry agar surface in a Petri dish. From colonies
developing, make agglutination and, if desired, cultural tests. It
is by immunity reactions that we identify cholera spirilla. The surface
moisture of plates is best dried by the filter-paper top.

The cholera colony is easily distinguished from the ordinary faecal bacterial colonies
by its transparent, bluish-gray, delicate character. It emulsifies with the greatest
ease. A practical, quick method is to make smears from suspicious colonies, stain
for one minute with dilute carbol fuchsin and if vibrios are present to make 2
vaseline rings on a single slide allowing ample space at one end for handling the
preparation safely. Inside of one ring deposit with a platinum loop a drop of salt
solution and inside the ring nearest the end which is to be held by fingers or forceps,
deposit a loopful of i to 500 or i to 1000 dilution of cholera serum. The emulsion
in the salt solution remains uniformly turbid and under a low power of the micro-
scope (% inch) shows a scintillating motility. The emulsion made into the drop
of serum quickly shows a curdy agglutination and upon examination with the
%-inch objective shows clumping and absence of motility. ^ Cover-glasses placed
over the 2 vaseline rings assist in the study of the preparation.

Cholera selective media are considered under "Culture Media."

CHAPTER X

STUDY AND IDENTIFICATION OF MOULDS

CLASSIFICATION OF THE FUNGI

Order

Suborder Family

Phycomycetes Zygomycetes Mucoracidae

Saccharo-
mycetidae

Gymnoas-
cees

Genus
Mucor
Rhizomucor

Ascomycetes

Is-'
(s.

Gymno-
ascidae

Carpoascees Perisporiacidae

Hyphomycetes

Species

M. corymbifer
M. mucedo
R. parasiticus
R. niger
cerevisiae
anginas
S. blanchardi
E. vuillemini
C. gilchristi

C. hominis

T. sabouraudi
T. tonsurans
T. violaceum
T. mentagro-
phytes
T. cruris
M. audouini
A. schoenleini

{P. montoyai
P. crustaceum
S. nidulans
A. fumigatus
A. concentricus
A. pictor
A. niger

D. bo vis
D. madurae
M. mycetomi
M. furfur

M. minutissi-
mus

M. albicans
T. giganteum
S. beurmanni

NOTE. — In many of the works on bacteriology considerable space is given to
the so-called Higher Bacteria. The organisms are chiefly considered under the
names Leptothrix or forms in which are found simple nonbranching threads, Clado-
thrix or thread-like forms with false branching and Streptothrix or forms showing
true branching. It is not practical to consider any separate group distinct from the
so-called Lower Bacteria on the one hand and the Fungi on the other.

136

Rhizopus
Saccharomyces

Endomyces
Cryptococcus

Trichophyton

Microsporum
Achorion

Penicillium
Sterigmatocystis

Aspergillus

Discomyces

Madurella
Malassezia
Microsporoides

Monilia

Trichosporum

Sporotrichum

FUNGI 137

The Thallophyta are plants in which there is no differentiation be-
tween root and stem.

The classes of Thallophyta which are of interest medically are i. the
Algae and 2. the Fungi.

The Algae contain chlorophyll. An exception to this is with the Schizophyta,
algae which include the Bacteriaceae or Schizomycetes and the Schizophycese or
Cyanophyceae. The bacteria do not contain chlorophyll and the Cyanophycese
or blue-green algae contain a blue pigment (phycocyanin) in addition to chlorophyll.
It is in their relation to bacteria that algae are important. Some authorities consider
the family Bacteriaceae as belonging to the order Cyanophyceae.

Diseases caused by fungi are known as mycoses.

Some include Lichenes as a separate class. These are really sym-
biotic organisms — Fungi parasitic on Algae.

The fungi do not possess chlorophyll. They are in their simplest forms ramifying
filaments called hyphae. The vegetative hyphae which intertwine in tangled threads,
as a support, are termed the mycelium, while those which project upward are called
the aerial hyphae and are the ones which bear the conidia or spores.

The aerial hypha which carries the fruiting organ encasing the conidia (sporan-
gium) is called the sporangiophore and the more or less rounded termination of this
hypha, which projects into the sporangium, is called the columella.

The hypha may be composed of one cell or of many cells separated by septa
(septate).

The orders of the class Fungi which are of interest medically are:
i. the Phy corny cetes; 2. the Ascomycetes; 3. the Hyphomycetes.

Phycomycetes. — These produce a copious network-like mycelium, which is non-
septate, and reproduce asexually by means of a sporangium, a case-like structure
borne on the clubbed extremity of an erect hypha (columella) and containing numer-
ous spores or, as in the case of the suborder Oomycetes, reproduction is by heter-
ogamy. (Dissimilar sexual cells — a smaller male, antheridium, and a larger female,
oogonium. By fertilization by antherozoids from the antheridium penetrating the
oosphere we have oospores.)

The suborder Zygomycetes reproduces either asexually (a sporangium filled with
spores) or by isogamy (two similar but sexually differentiated cells conjugate and
form on fusion a zygospore).

Belonging to this suborder we have four families, only one of which, the Mucor-
acidae, is of importance medically. In this family we have three genera: Mucor,
without rhizoids; Rhhopus, with rhizoids and unb ranched aerial hyphae and,
Rhizomucor, with rhizoids and ramified mycelium.

Under anaerobic conditions the non-septate mycelium of these fungi may break up
into short septa resembling yeasts.

Two species of Mucor are of pathogenic importance.

STUDY AND IDENTIFICATION OF MOULDS

i. Mucor mucedo and 2. Mucor corymbifer. These moulds develop especially in
external cavities as nasopharynx and external ear.

Pulmonary and generalized infections have also been reported. The pathogenic
species have smaller spores and grow best at 37°C. The thick, coarse, cotton-like
mould seen on horse manure is a Mucor. The sporangium, the organ of fructifica-
tion, contains the spores within its interior. The M. mucedo has thick silver-gray
mycelium, with large sporangia, 150/1 in diameter, containing oval spores, 5 X QM-
The M . corymbifer, which has been reported from a generalized infection, considered
as typhoid, shows a snow-white mycelium. The sporangia are 20 to 40ju and the
spore about 3/1 in diameter.

Rhizomucor parasiticus has been reported from the sputum of a
woman with a condition resembling phthisis.

Rhizopus niger has a columella which becomes distorted into a mushroom shape
after the spores have been discharged from the sporangium. This mould has been
considered as the cause of a mycosis of the tongue.

>y

FIG. 40: — Yeast cells. Saccharomyces cerevisice. (Coplin.)

Ascomycetes. — In this order are included many of the parasitic
moulds. The most distinctive characteristic is the formation of asco-
spores in an ascus (little sac).

It is an enlargec! extremity of a hyphal branch in which a definite number of spores,
usually eight, is formed. The ascus usually ruptures at its tip. Other members of
the order are formed from hyphae by the separation of cells in succession from the
free cells. The mycelium is septate.

The order is divided into those with naked asci (Gymnoascees) and
those having a perithecium or investing layer about the ascus (Car-
poascees).

YEASTS

139

Belonging to the suborder Gymnoascees we have i. the family of Saccharomyce-
tidas, which reproduce by budding and in which the asci are without any semblance
of a sheath, and 2. a family in which there is an indication of the formation of a peri-
thecium — this may be termed the Gymnoascidae family.

SACCHAROMYCETID.E. — There are three genera: Saccharomyces, Endomyces, and
Cryptococcus.

Saccharomyces. — These reproduce by budding, have ascopores and no mycelial-
like threads.

S. eeremsia. — This is the ordinary yeast fungus. Used at times as an antiseptic.
It is also used in treatment of beriberi as it is very rich in vitamines.
S. angina. — Found in a case of angina.

S. blanchardi. — Found in a jelly-like tumor mass of the abdomen. The budding
cells varied from 2 to 20^1. Probably identical with S. tumefaciens, reported
as the cause of a subcutaneous tumor about region of Scarpa's triangle.
Endomyces. — Forms spores in the interior of filaments, or by ascus formation or by
chlamydospores (resistant spore-like structures with a thick membrane which
project from the extremities or sides of the hyphae as bud-like structures).
E. vuillemini. — One of the organisms of thrush. It produces a false membrane,
especially on buccal surfaces, which is easily detached and beneath which the
mucosa is intact. Grows only in acid media. Hence propriety of alkaline
treatment. Some authorities consider the genus Endomyces as the same as
Monilia or Oidium.

Cryptococcus. — Reproduces by budding, but ascospore formation not observed.
Not a well-recognized genus. The diseases caused by it are termed blasto-
mycoses.

C. gilchristi. — The cells are about i6ju in diameter and have a thick, double con-
toured membrane. They reproduce by budding. The skin lesions resemble
various infectious granulomata and diagnosis rests on the finding of budding
or sporulating cells. It may invade internal organs. Original cultures are
obtained with some difficulty and then best with Lender's serum. Subcultures
grow readily. Potato is a good medium and on it we may have both mycelial
and yeast-like growth. Guinea-pigs can be inoculated subcutaneously. A mould,
somewhat similar, is the Coccidioides immitis of Ophuls. This has a mycelial
growth in tissues, this distinguishing it from the former fungus. The large
cells frequently show yeast-like bodies within, hence the characteristic of en-
dogenous spore formation. The large round encapsulated cells of C. gilchristi
or C. hominis do not show contained spores. The infection frequently becomes
generalized. The small bodies, about 3/1, in the Molluscum contagiosum cells
are thought by some to be yeasts. They are more probably artefacts.
Plimmer's bodies in cancer cells belong in this group. They also are probably
other than parasites.

C. lingua pilose.— This is a more or less elongated yeast-like organism and sup-
posed to be the cause of black tongue, a benign affection of the lingual papillae.

GYMNOASCIDAE. — Belonging to the family Gymnoascidae we have
the genera Trichophyton, Microsporum, Achorion, Endodermophyton
and Epidermophyton.

140

STUDY AND IDENTIFICATION OF MOULDS

Trichophyton. — The fungi of the genus Trichophyton are generally known as
the large-spored ringworms. The spores are in chains and may be inside the hair
or both outside and inside. Many of them are of animal origin, especially from
the horse and the cat. The spores are from 5 to i5/z.

The mycelium is greatly segmented, shows simple or dichotomous branching,
and produces spores within the mycelium.

T. tonsurans. — Gives a crater-like culture with fine marginal rays. Fungus wholly
inside the hair. Causes most of the large-spored scalp ringworms and many
body cases.

10.

FIG. 41. — More common fungi, i, Culture of Achorion schoenleini (favus);
2, culture of Trichophyton tonsurans; 3, culture of Trichophyton sabouraudi; 4,
sporangium of Aspergillus; 5, culture of Trichophyton mcntagrophytes; 6, culture
of Microsporum audouini; 7, mycelium and spores of Malassezia furfur; 8, Crypto-
coccus gilchristi; 9, A and B, sporangium and mycelium of Mucor corymbifer; 10,
Penicillium; n, Saccharomyces tumefaciens; 12, Discomyces bovis.

It is the T. megalosporum endothrix of Sabouraud.

The short, diseased fragmented hairs are mouldy looking. The spores are 5 to 6
microns.

T. sabouraudi. — Has a heaped-up festooned sort of culture. There is a similar
fungus with a violet culture. These cause some of the scalp and beard ring-
worms.

It is easily dissociated in a 2 or 3% solution of caustic potash while T. tonsurans
is hard to break up. The hairs are broken off close to the skin, hence "black dotted
ringworm."

TINEA IMBRICATA

141

T. mentagrophytes.—This is the T. megalsporon endoectothrix of Sabouraud. The
external spores are in chains or in short mycelial threads, not mosaics of spores,
and are of very unequal size (3 to 15 microns).

The internal spores are scarce and are from 5 to 6 microns in diameter. To exam-
ine pull out downy hairs from the periphery of the lesion rather than the dead
central ones. There are varieties from horse, cat, and bird. The lesions are more
inflammatory than those of the endothrix class. Most of the beard and body ring-
worms belong to this group— very few scalp cases. The lesions are often of a pus-
tular type. The cultures are finely rayed.

Some give yellow cultures, others white and one derived from birds a rose-colored
culture.

Microsporum audouini. — This is the so-called small-spored ring-
worm and is a very common and highly contagious affection of the scalp
in children in England and France; less so in other countries.

It is almost never seen in the tropics. It almost exclusively affects the hairy
scalp. The spores are 2 to 3/4 in diameter. The broken stump of the hair is char-
acteristic. The fungus is packed as a mosaic of spores, forming a white sheath,
chiefly on the outside of the hairs. It gives a downy-white culture.

Achorion schoenleini is the cause of favus. The cultures are rather
wrinkled. It is characterized by the scutulum or favus cup. This is a
sulphur-yellow pea-sized cup with a central lusterless hair. Affected
hairs may not show a cup. Favus is not so contagious as ringworm.
It chiefly affects the hairy scalp, but may also invade the nails and even
the body.

Microscopical examination shows great irregularity of spores and mycelium, the
latter being irregularly disposed and of varying thickness and length and wavy
instead of straight as in Trichophyton. There is also the greatest irregularity in
the refractile favus spores — they are gnarled and bizarre shaped, in contrast to the
regular ovals or spheres of the ringworm fungus. Cultures show ridges or con-
volutions.

Endodermophyton concentricum. Castellani considers this as the
causative fungus of tinea imbricata rather than Aspergillus concentricus .

It was formerly supposed that the causative fungus was Aspergillus concentricus
but Castellani has demonstrated that fungi of this genus, when present, are merely
accidental. He has isolated in cultures what he considers the causative fungus,
Endodermophyton concentricum. He treated scales for ten minutes with absolute
alcohol and then placed single scales in a series of tubes of maltose bouillon. The
fungus grows between the rete malpighii and the external epidermal layers forming
a network of mycelial threads, about 3 microns broad.

Another fungus cultured from tinea imbricata scales is Endodermophyton indicum.
Inoculation of this organism in pure culture produced the disease.

The characteristics of the genus Endodermophyton are the growth of a mycelial

142 STUDY AND IDENTIFICATION OF MOULDS

network between the rete malpighii and the superficial epidermal layers. In cul-
tures only mycelial filaments are found, there are no conidia bearing hyphae.
The fungus is also called Trichophyton concentricum.

Epidermophyton cruris. (See Microsporoides minutissimus, page 145) .
Under the name "dhobie itch" this fungus affection is probably
better known to Europeans than any other tropical skin disease.

This name dhobie or washerman's itch has been given on account of associating
it with the infection of the underclothing while being washed in the pools or streams
along with the garments of those who have this skin disease. This, like every other
widespread view, has probably some foundation but cannot be verified. It is the
eczema marginatum of Hebra.

This affection is caused by various species of Epidermophyton. This genus
differs from Trichophyton in that it never invades the hair or hair follicles.

The species which have been more frequently reported are Epidermophyton crttris,
E. perneti and E. rubrum. The mycelium is about 4 microns broad and the spores
about 5 or 6 microns. All of these fungi can be cultured on Sabouraud's maltose
agar, growth appearing in about a week, except E. perneti, which grows more rapidly.

IN THE SUBORDER CARPOASCEES we have to consider the family
Perisporiacidae. In this family the asci are completely inclosed by the
investing membrane, the perithecium. When this rots the spores are
set free. There are three genera of interest, Penicillium, Aspergillus
and Sterigmatocystis.

In Penicillium we have vertical branches with strings of conidia. In Aspergillus
these conidia arise from a globular termination of the hypha.

Penicillium. — While Penicillium does at times form perithecia, yet they char-
acteristically show chains of spores. The common P. glaucum resembles a hand
with terminal beads, more than the hair pencil, from which the name is derived.

P. crustaceum. — Is the common blue-green mould. It has been deemed patho-
genic in cases of chronic catarrh of the Eustachian tube and in gastric hyper-
acidity.

P. montoyai. — Cause of violet pinta.

Aspergillus. — These have sterigmata carrying chains of spores, these sterigmata
being little processes projecting out from the knob-like termination of the aerial
hypha (columella). Of the pathogenic Aspergilli we have:

1. A. fumigatus. — This has been considered as the cause of pellagra. A pul-
monary mycosis resembling phthisis may be due to this species.

2. A repens. — This has been found in the auditory canal and may produce a
false membrane.

3. A. flavus. — This has been found in the discharges of chronic ear diseases.

4. A. concentricus. — Formerly considered as the cause of an important tropical
ringworm, tinea imbricata. The scales are dry, like pieces of tissue-paper.
There are generally about four rings which do not heal in the center. General
appearance is that of watered silk. There are no inflammatory lesions. Com-

PINT A 143

mon in Malay peninsula. Also found in some parts of the Philippines and in
China. Some authorities consider the fungus to be a Trichophyton.
Castellani claims that the cause is a fungus which develops between the stratum
corneum and the deeper layers of the epidermis, Endodermophyton concentricum.
It differs from the achorions in not invading hair follicles. (See page 141.)

5. A. pictor. — This is the cause of a skin affection of Central America (Pinta).
In the affection colored spots appear on the skin, chiefly on face, forearms, and
chest. The disease is attended with a mangy odor. Spots are of various
colors; if the superficial epithelium is affected we have a dark violet color.
Deeper involvement gives red spots.

Other names for the disease are caraate and mal de los pintos. At first it was
thought that the different colors shown by the eruption were due to varying depths
of the proliferating fungi in the skin layers but it is now known that the explanation
is in a variety of species in the different types of pinta.

The pure violet pinta is caused by Aspergillus pictor, while the grayish- violet one
is due to Penicillium montoyai. A species of Monilia causes the white variety and
different species of Montoyella a black and a red variety respectively. The genus
Montoyella is stated by Castellani to have both slender and thick mycelial threads,
from the thicker of which spring delicate hyphae terminating in pear-shaped conidia.
Material scraped from the lesions and mounted in liquor potassae shows the
fructification terminations characteristic of Aspergillus or Penicillium in the violet
or gray- violet varieties while the white, black and red ones only show mycelial threads
and scattered spores. These pinta species of fungi can be cultivated on Sabouraud's
medium.

Montoya thinks that the pinta fungi lead a saprophytic existence in the waters of
mines or other places with a constant high temperature, and states that he has
obtained pure cultures from such sources.

Sterigmatocystis. — This genus has chains of conidia, similiar to those
of Penicillium, but these are borne on other short chains, which arise
from the clubbed aerial hyphae (conidiophores). These are called
secondary and primary sterigmata, respectively.

S. nidulans. — This fungus has been found in cases of otomycosis as well as in the
white granules of mycetoma.

Hyphomycetes.— In this order are grouped certain genera which
cannot properly be assigned to any of the other orders. They are also
designated Fungi Imperfecti, for the reason that the fruiting bodies
characteristic of the other orders have not been satisfactorily observed.

Discomyces bo-vis — This is the well-known ray fungus, the cause of actinomycosis. In
man it is at times found in chronic suppurative conditions attended with much
granulation tissue. Such pus may show small yellow-gray granules about the
size of a pin's head. When spread out between two slides the central portion
shows a network of mycelium with bulbous thread-like rays going to the periph-
ery. The "clubs" at the periphery are degenerate structures and do not stain
by Gram. The central mycelium is Gram-positive. This mould is essentially
an anaerobe and should be cultivated in a deep glucose agar stab. It may also

144

STUDY AND IDENTIFICATION OF MOULDS

be cultivated in bouillon. In this it grows at bottom. Growth is dry and
chalky. In diagnosis look for the little granules. Curetting of the sinuses
may give the "ray fungus" when they are not found free in the pus.
Discomyces madura. — This is a ray fungus found in the yellow " fish-roe" granules
of madura foot. The disease is caused by the penetration of certain species of
fungi into the tissues of the foot, although rarely the hand or some other part of
the body may be affected. These species of fungus develop in granulomatous
areas from which sinuses lead to the surface of the foot, in the discharges from
which are found small granules resembling those found in the discharges from
actinomycosis lesions.

££i?u^°n (ft\ .Cndomyces vuillemini

(Q)(monilia or Oidium albicans)

Cpidermophyton /y\ Coccidipid&s
cruria V« I immitta

Cryptococcujs

linyuaa-piloaoe. QA Trichosporum giyanteum

T. imbricata (aide view)
mycetorni

homini, ££%%

FIG. 42. — Important tropical fungi.

As a rule only one kind of fungus is found in a single case. The most common
infection is that due to Discomyces madura (Nocardia madura) which is the fungus
of the fish-roe like granules of the pale or white variety of mycetoma. These like
the fungus of actinomycosis, Discomyces bovis, show a felted mycelium in the center
and peripheral club-like structures. The granules are yellowish white and vary in
size from a pin's head to a small pea. The mycelial threads are very narrow, i to
iH microns. It grows aerobically and the cultures show slender mycelial threads
which are Gram-positive. This is the organism of Carter's white mycetoma.

Other species of the pale, white or ochroid group of mycetoma fungi are Indiella
mansoni (Brumpt's white mycetoma) Nocardia asteroides (Musgrave and Clegg's
white mycetoma) Sterigmatocystis nidulans (Nicolle's white mycetoma) and several
others.

MONILIA AND SPRUE 145

The cases caused by the black varieties are more rare and are characterized by the
presence in the discharge from the sinuses of black gunpowder-like grains.

These hard, brittle, irregular grains are caused by various species of fungi of
which the best known is Carter's black mycetoma (Madurella mycetomi). This
species was cultured by Wright and first shows a grayish growth, later becoming
black. Other black varieties of mycetoma are due to various other fungi. Bouf-
fard's black variety is caused by Aspergillus bou/ardi. DeBeurmann's black myce-
toma has as cause Sporotrichum beurmanni.

Besides the white and black varieties we also have a red variety of mycetoma.
The fungus grains are quite small and reddish in color. It is not an uncommon
infection in certain parts of Africa, as Senegal. The cause is Nocardia pelletieri.
Discomyces carougeani has been reported as the cause of juxta-articular nodes,

but Breinl has been unable to verify the finding.

M alassezia furfur. — This is the fungus of tinea versicolor. It is common both in
temperate and in tropical climates. It is characterized by dirty yellow spots
about covered parts of the body. Scrapings shows a profusion of mycelial
threads and interspersed spores. It is very difficult to cultivate. The organism
usually termed the bottle bacillus is really a fungus having the characteristics
of the genus Malassezia. It is thought to be the cause of pityriasis of the
scalp.

Microsporoides minutissimus. — This is generally considered as the cause of Ery-
thrasma or dhobie itch, a very common intertrigo of the tropics. It is char-
acterized by its narrow mycelium and small spores. Various fungi are found
in this affection. Castellani considers the chief cause of dhobie itch to be a
trichophyton. Epidermo phyton cruris.

Clinically this affection shows festooned areas of a bright red color which tend
to clear up in the center becoming fawn color. As a result of the intolerable itching
and scratching the affection tends to spread from its favorite sites — the inner sur-
faces of the thighs and the scrotum. The spores and mycelium are abundant at
the onset but later, one may not find any evidence of the mould. In some of the
rapidly spreading cases I have found a symbiosis of fungus and coccus, the bacterial
elements lying packed in aggregations scattered through the mycelial ground work.
Culturally these cocci were S. pyogenes aureus. (See page 142.)

Monilia albicans (Oidium albicans). — Castellani separates Monilia
from Endomyces in that it does not show the asci and internal spores
of the latter. In cultures it gives budding yeast-like growths and
mycelial threads. On Sabouraud's medium it gives a whitish growth.
It slowly liquefies gelatin and blood-serum and, after acidifying, clots
milk. It is recognized as the organism of thrush. Bahr found this
fungus in the deep layers of the tongue as well as in the cesophageal
and intestinal coatings of sprue.

Ashford is convinced that a species of Monilia which he is sure is distinct from
M . albicans, is the cause of sprue. He states that this fungus is common in the
bread of Porto Rico. He has recovered the organism from sprue lesions and has

146

STUDY AND IDENTIFICATION OF MOULDS

produced a monilia septicemia in rabbits inoculated with the sprue fungus. This

fungus is quite pathogenic when first isolated from sprue lesions.

In internal organs the mycotic areas are not associated with pus formation. On

two occasions he has produced stomatitis in animals by feeding experiments.

Monilia tropicalis. — Castellani has reported this fungus as the cause of a broncho-
mycosis. It does not coagulate milk nor liquefy gelatin. The growth on
Sabouraud's medium is mainly of yeast-like cells.

M . Candida. This fungus was found in white patches on tongue of
a child. The conidia are 7 to 8 microns and the mycelium i to ij^ mic-
rons in diameter.

Boggs has recently isolated a Monilia which he considers as closely related to
M . Candida. The patient was at first thought to have a mammary carcinoma with
axillary gland metastases. Later on there was a severe cough with abundant red-

FIG. 43. — Thrush fungus. (Kolle and Wassermann.)

dish-gray sputum which showed mycelium and yeast-like cells. Cultures on glucose
agar, potato, etc., at 37°C. and at room temperature gave a moist glistening
whitish growth which, when examined, showed only yeast-like cells, no mycelial
growth. Hyphae only showed in later cultures.

There was a fair growth in milk with after three or four weeks an alkaline reac-
tion and firm coagulum. Slight acid production in glucose but none in lactose,
saccharose or mannite.

Mycelial growth was more rapid in anaerobic cultures than in aerobic ones.
Boggs notes that his Monilia is morphologically indistinguishable from the Monilia
of sprue. The buccal mucosa of this case did not show any abnormalities.
Trichosporum giganteum. — This is the cause of a disease of the hairs, known in

Columbia as "Piedra," so called from the small gritty-like masses along the

length of the hair. These spores are arranged like mosaics about the hair.
Sporotrichum beurmanni. — This fungus has a narrow mycelium (2/1) and branches

in all directions. The spores appear as little grape-like clusters of oval spores

SPOROTRICHOSIS 147

(3 to SM) at the end of a filament. It is readily cultivated, showing as a small

white growth about the eighth day.

The fungus of sporotrichosis develops in tissue by budding, not showing the
mycelial growth seen in artificial cultures. Potato makes a good medium and often
such cultures show pigmentation.

This mould produces indolent, glistening, subcutaneous tumors which are pain-
less. They may ulcerate and give off a brownish discharge. They resemble tuber-
culous or syphilitic lesions.

Certain organisms which resemble both moulds and bacteria, having branching
filamentous forms and at the same time having a spore-like method of reproduction,
are known under the names Streptothrix or better Nocardia. It is chiefly in various
pathological processes of the lungs that they have been observed, but in addition
they have been noted in brain, glands, kidney and subcutaneous tissue.

The infections are most likely to be confused with phthisis and glanders. The
organisms are easily cultivated and in staining reactions are midway between T. B.
and Actinomycosis.

Castellan! uses the generic name Nocardia for Discomyces.

DIAGNOSIS or FUNGI

The most expeditious way to examine for fungi is to treat the scales
or hairs with a 10% solution of caustic potash or soda. Then crush
between two slides; heat moderately over the flame and examine.

Tribondeau's method is to treat the scales with ether, then with alcohol, and
finally with water. Next put the sediment (it is convenient to use a centrifuge)
in a drop of caustic soda solution. Cover with a cover-glass, and after the prepara-
tion has stood about an hour run glycerine under the cover-glass.

A very satisfactory method is to scrape the scales with a small scalpel, and smear
out the material so obtained in a loopful of white of egg or blood-serum on a glass
slide. By scraping vigorously the serum may be obtained from the patient. After
the smear has dried, treat it with alcohol and ether to get rid of the fat. It may then
be stained with Wright's stain or by Gram's method. The ordinary Gram method
may be used or the decolorizing may be done with aniline oil, observing the decolori-
zation under the low power of the microscope.

Yeasts are best examined in hanging drop on the plain slide with vaselined cell,
as given under Blood.

An excellent way to examine moulds is to seize some of the projecting sporangia
from the surface of a plate with forceps and mount in liquid petrolatum. I have
found that moulds in scales from skin or infecting various mites or insects will show
a growth in this medium when mounted on a slide and covered with a cover-glass.

CULTIVATION or FUNGI

Moulds grow well on media with an acid reaction, so that by adjust-
ing the reaction to +2% or even higher, we permit of the growth of the
fungi, but inhibit bacterial development.

148

STUDY AND IDENTIFICATION OF MOULDS

Glycerine agar, bread paste, or potato media are all suitable, but the best medium
is that of Sabouraud:

Maltose, 4 . o grams.

Peptone, i . o gram.

Agar, i . 5 grams.

Water, 100.0 c.c.

Make the reaction about +2.

Before inoculating media with moulds, some recommend placing the material in
60% alcohol for one or two hours to kill the bacteria. The moulds withstand such
treatment.

In cultivating moulds it is best to use small Erlenmeyer flasks, containing about
Y± in. of media on the bottom, for the development of the colonies. In order to
separate the mould we may take the hair or scales on a sterile slide and cut them
into small fragments with a sterile knife. Then moisten a platinum loop from
the surface of an agar slant, touch a fragment with the loop, and when it adheres
transfer it to the agar slant. Make four or five inoculations on the surface and from
suitable growth, after four to seven days, inoculate the medium in the Erlenmeyer
flask. Esmarch roll cultures are better than flask ones.

Plauth recommends receiving the mould material between two sterile glass slides.
Seal the edge of the slide with wax and place the preparation in a moist chamber for
four to seven days. From developing fungus growth inoculate the medium in the
Erlenmeyer flask. A Petri dish containing several layers of thoroughly moistened
filter-paper in top and bottom makes a satisfactory moist chamber.

For the study of the morphology of Monilia in cultures, Boggs used
stab cultures of 15% gelatin. The growth in the tube was hardened
in 10% formalin, the glass cracked off and sections of the gelatin
column cut across at any desired level. These blocks were sectioned
with the freezing microtome; stained in dilute aqueous fuchsin (i to
30) for several hours; then differentiated in saturated solution of citric
acid until nearly decolorized. The sections were floated on slides, air
dried without blotting, cleared in xylol and mounted in balsam.

For staining fungi in sections of tissue Busse recommends the folio wing method:
i. Haematoxylin, 10 to 15 minutes, then wash in tap water. 2. Carbol fuchsin
(i to 20) 30 minutes or over night. Decolorize in alcohol for a few minutes
then through absolute alcohol and xylol to mount in balsam. The moulds are red.

CHAPTER XI
BACTERIOLOGY OF WATER, AIR, MILK, ETC.

BACTERIOLOGICAL EXAMINATION OF WATER

WHILE in a chemical examination as to the character of a water
there are certain relations between the free and albuminoid ammonias,
nitrates, nitrites, chlorides, etc., which indicate the probable animal as
against vegetable nature of the organic matter present, yet it is a more
or less presumptive evidence. In a bacteriological examination of
water the finding of the colon bacillus may from a practical standpoint
be considered as positive evidence of human faecal contamination.
Theoretically, the possibility of organisms being present corresponding
culturally to B. coli and derived from cereals is to be considered. Also
the faeces of animals contain an organism which cannot be differentiated
from the colon bacillus.

In detecting sewage contamination in water to which varying amounts of sewage
had been added, it was found that the bacterial tests were from 10 to 100 times
more delicate than the chemical ones.

As showing sewage contamination of water, the presence of the B. coli has been
generally accepted as the most satisfactory indication. The English authorities
consider sewage streptococci and the spore-bearing B. enter itidis sporo genes as of
value as indicators as well as the B. coli — the presence of sewage streptococci indicat-
ing very recent sewage contamination and that of the B. enteritidis spcro genes, in the
absence of streptococci and colon bacilli, as evidence of sewage contamination at some
period more or less remote.

In the United States the colon bacillus alone is considered the indi-
cator of sewage contamination, and all tests, presumptive or positive,
are based on the presence of this organism.

It is not the finding of the colon bacillus but rather the question of
its relative abundance that is involved in a water analysis. Thus the
finding of one colon bacillus in 50 c.c. of water would not have weight as
showing contamination, but the presence rather constantly of the colon
bacillus in i c.c. or less makes contamination of a water supply probable.

In collecting samples of water for bacteriological examination, the following points
should be considered:

149

150 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

i. The bottles, which should have a capacity of from 25 to 100 c.c., should be
sterile. Sterilization may be effected by heat or by rinsing with a little sulphuric
acid and subsequently washing out thoroughly with the suspected water before col-
lection. The utmost care must be exercised that the fingers do not come in contact
with the glass stopper of the neck of the bottle while filling it. If the specimen is
to be sent some distance, it should be packed in ice to prevent bacterial development.
Frankland states that a count of 1000 became 6000 in six hours and 48,000 in forty-
eight hours. In water packed in ice for a considerable time, however, the bacterial
count may diminish.

2. If collecting from city water supplies, secure the sample direct from the mains
and let the water run from the tap a.few minutes before collection. If the water be
taken from a pond, stream, or cistern, be sure that the specimen comes from at least
10 inches below the surface. As sedimentation is the most important method in
self-purification of rivers and ponds, it will be understood that any stirring up of the
mud on the bottom will enormously increase a bacterial count.

Quantitative Bacteriological Examination

i. Deliver definite quantities of the water to be examined into tubes of liquefied
gelatin or agar and plate out the same in a series of Petri dishes.

A more practical method is to deliver the water from the graduated pipette into
the empty sterile dish. The water should be deposited in the center of the plate and
the melted gelatin or agar poured directly on the water and then, carefully tilting to
and fro, mix the water and the media. One set of plates should be of gelatin and
incubated at room temperature; a similar set should be of lactose litmus agar and
incubated at 38°C. If the water is highly contaminated, it is necessary to dilute
it; thus, with river water, which may contain from 2000 to 10,000 bacteria per c.c.,
a dilution of i to 100 would be desirable.

Ordinarily it will be sufficient to deliver from a sterile graduated pipette 0.2, 0.3,
and 0.5 c.c. of the water in each of two sets of plates: one set for gelatin, the other
for agar.

When gelatin is not at hand or convenient to work with, the gelatin plates may
be replaced by others of lactose litmus agar for incubation at room temperature.
After twenty-four hours at 38°C. or forty-eight hours at 2o°C., the count should be
made.

Example. — Forty colonies were counted on the gelatin plate containing 0.2 c.c.
(^5) of the water. The number of organisms would be 200 per c.c. Ten colonies
were counted on the agar plate containing 0.2 c.c. and incubated at 38°C. Number
of bacteria developing at body temperature equals 50 per c.c.

There is no strict standard as to the number of bacteria a water should contain per
c.c. Koch's standard of 100 colonies per c.c. is generally given. It is by the
qualitative rather than the quantitative analysis that one should judge a water.

If there should be very many colonies on a plate, the surface can be marked off
into segments with a -blue pencil. If very numerous, cut out of a piece of paper a
space equal to i sq. cm. By counting the number of colonies inclosed in this
space at different parts of the plate, we can strike an average for each space of i
sq. cm. To find the number of such spaces contained in the plate, multiply the

BACTERIOLOGY OF WATER 151

square of the radius of the plate by 3.1416. Then multiply this number by the
average per square centimeter, and we have the total number of colonies on the
plate. This is the principle of the Jeffers disc.

The relative proportion between the bacterial count at 2o°C. and that at 38°C.
is of great importance from a qualitative standpoint, as will be seen later.

2. Deliver into a series of Durham fermentation tubes containing glucose bouillon
and into another series containing lactose bouillon varying definite amounts of the
water to be examined. In tubes showing the presence of gas in both glucose and
lactose bouillon the evidence is presumptive that the colon bacillus is present. For
the positive demonstration plates must be made from such tubes as show gas.

It is sufficient to deliver from graduated pipettes in each series quantities of
water varying in amount from o.i c.c. to 10 c.c. In our laboratory we inoculate
with o.i c.c., 0.2 c.c., 0.5 c.c., i c.c. and 10 c.c. of the suspected water. If the o.i c.c.
tubes show gas, we have reason to assume that the water contained at least 10 colon
bacilli per c.c. If only the 10 c.c. tubes showed gas — those with less amounts not
having gas — we would be in a position to state that the water contained the colon
bacillus in quantities of 10 c.c., but not in quantities of i c.c. or less. Many authori-
ties regard water as suspicious only when the colon bacillus is present in quantities
of 10 c.c. or less; waters of good quality frequently showing the presence of the colon
bacillus in quantities of 100 to 500 c.c.

It is generally accepted that if a water shows the presence of the
colon bacillus in quantities of i c.c. or less, it should be regarded as
suspicious.

At the present time the medium that gives the least source of error in carrying
out the quantitative presumptive tests is the lactose bile. It is made by adding
i % of the lactose and i % of peptone to ox bile, and fermentation tubes of the media
showing gas may be considered as very probably containing the colon bacillus. The
percentage of error with this method is reported to be only 11%, while with glucose
fermentation tubes the error is more than 50%. Gas formation is usually shown in
forty-eight hours, but it is advisable to continue the incubation for seventy-two hours.
These presumptive tests are chiefly of value in highly contaminated waters. Even
with this method plates should be made.

3. As the colon and sewage streptococci ferment lactose with the production of acid
and hence produce pink colonies on lactose litmus agar, much information can be
obtained from the proportion existing between the number of pink colonies and
those not having such a color. Waters of fair degree of purity rarely give any pink
colonies.

Qualitative Bacteriological Examination

General Considerations.— In some countries the proportion of
liquefying to nonliquefying colonies on gelatin plates is considered of
importance. Certain sewage organisms belonging to the proteus and
cloaca groups liquefy gelatin; consequently, if the proportion of liquefy-
ing to nonliquefying be greater than as i to 10, the water is considered

152 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

suspicious. The test is not considered by American authorities as of
any particular value.

The American Public Health Association recognizes the importance of the informa-
tion obtained from a comparison of the number of organisms developing at 38°C.
and those developing at 20°C. Bacteria whose normal habitat is the intestinal canal
naturally develop well at body temperature, while normal water bacteria prefer the
average temperature of the water in rivers and lakes. Consequently when the
number of organisms developing at 38°C. at all approximates the number developing
at 2o°C., there is a strong suspicion that sewage organisms may be present. Normal
waters give proportions of i to 25 or i to 50, while in sewage contaminated waters the
proportion may be as i to 4 or less.

In addition, the appearance of pink colonies on the lactose litmus
agar is of great assistance in judging of a water. Both sewage strepto-
cocci and the colon bacillus give pink colonies — those of the streptococci
are smaller and more vermilion in color. Microscopic examination
will differentiate the cocci from the bacilli. It is well to bear in mind
that the pink colonies after twenty-four hours may turn blue in forty-
eight hours from the development of ammonia and amines. Conse-
quently the lactose litmus agar plates should be studied after twenty-
four hours.

A good water supply will rarely show a pink colony, while in a sewage-contami-
nated one the pink colonies will probably predominate.

A commission composed of eminent American bacteriologists and
sanitarians have recommended the following as maximum limits of
bacteriological impurity :

1. The total number of bacteria developing on standard agar plates,
incubated twenty-four hours at 37°C.; shall not exceed 100 per c.c.
Provided that the estimate shall be made from not less than two plates,
showing such numbers and distribution of colonies as to indicate that
the estimate is reliable and accurate.

2. Not more than one out of five 10 c.c. portions of any sample
examined shall show the presence of organisms of the Bacillus coli
group when tested as follows:

(a) Five 10 c.c. portions of each sample tested shall be planted, each in a fermenta-
tion tube containing not less than 30 c.c. of lactose peptone broth. These shall be
incubated forty-eight hours at 37°C. and observed to note gas formation.

(6) From each tube showing gas, more than 5% of the closed arm of fermenta-
tion tube, plates shall be made, after forty-eight hours' incubation, upon lactose
litmus agar or Endo's medium.

(c) When plate colonies resembling B. coli develop upon either of these plate

THE COLON GROUP 153

media within twenty-four hours, a well-isolated characteristic colony shall be
fished and transplanted into a lactose-broth fermentation tube, which shall be
incubated at 37°C. for forty-eight hours.

For the purpose of enforcing any regulations which may be based upon these
recommendations the following may be considered sufficient evidence of the presence
of organisms of the Bacillus coli group.

F*V^.— Formation of gas in fermentation tube containing original sample of water.

Second. — Development of acid-forming colonies on lactose litmus agar plates or
bright red colonies on Endo's medium plates, when plates are prepared as directed
above under (6).

Third. — The formation of gas, occupying 10% or more of closed arm of the
fermentation tube, in lactose peptone broth fermentation tube, inoculated with
colony fished from twenty-four-hour lactose litmus agar or Endo's medium plate.

These steps are selected with reference to demonstrating the presence in the sample
examined of aerobic lactose-fermentating organisms.

3. It is recommended, as a routine procedure, that in addition to
five 10 c.c. portions, one i c.c. portion, and one o.i c.c. portion of each
sample examined be planted in a lactose peptone broth fermentation
tube, in order to demonstrate more fully the extent of pollution in
grossly polluted samples.

The members of the B. coli group are separated by the American
Public Health Association according to fermentation activities as

follows :

B. Coli Group.
Dextrose +
Lactose +

Dulcite + Dulcite —

B. communior B. aerogenes

B. communis B. acidi-lacti

i i

Saccharose + Saccharose - Saccharose + Saccharose -

B. communior B. communis B. aerogenes B. acidi-lactici.

Further divisions are made by the use of mannite and raffinose, giving varieties of
the above four species.

Dulcite, like mannite, is a hexatomic alcohol. It is isomeric with mannite and is
present in Madagascar manna.

Raffinose is a trisaccharide; maltose, lactose and saccharose are disaccharides, while
dextrose and l<evulose (hexoses) arabinose and xylose (pentoses) are monosaccharides.

The diagnostic characteristics considered important by the Ameri-
can authorities in reporting the colon bacillus (recently designated
excretal colon bacillus) are:

154 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

1. Typical morphology, nonsporing bacillus, relatively small and often quite thick.

2. Motility in young broth cultures. (This is at times unsatisfactory, as some
strains of the colon bacillus do not show it even in young bouillon cultures).

3. Gas formula in dextrose broth. Of about 50% of gas produced, one-third
should be absorbed by a 2% solution of sodium hydrate (CO2). The remaining gas
is hydrogen. (Later views indicate that the gas formula is exceedingly variable and
should not be depended upon. To carry out this test one fills the bulb of a fermenta-
tion tube with the caustic soda solution, holding the thumb over the opening or with
a rubber stopper, the bouillon culture and the soda solution are mixed by tilting the
fermentation tube to and fro. The total amount of gas is first recorded and then
that remaining after the CO2 has been absorbed is reported as hydrogen.)

4. Nonliquefaction of gelatin.

5. Fermentation of lactose with gas production.

6. Indol production.

7. Reduction of nitrates to nitrites.

To these may be added the acidifying and coagulation of litmus milk without
subsequent digestion of the casein. The production of gas and fluorescence in glu-
cose neutral red bouillon is also a very constant function of the colon bacillus. B.
coli aerogenes is similar to B. coli with the exception of nonmotility, formation of gas
in starch media (bubbles on potato slant) and frequent lack of indol production.
It is often, especially in milk cultures, provided with a capsule and rarely forms
chains. It is a member of the Friedlander group but differs from the typical pneu-
mobacillus by producing acid and gas in lactose broth and by its coagulation of
milk.

B. coli anaerogenes is also similar to B. coli but does not produce gas in glucose and
lactose. This latter organism is not usually recognized by American authorities
but I have found on Endo plates an organism showing the red colony with metallic
luster which failed to produce gas in either glucose or lactose.

NOTE. — The reduction of neutral red with a greenish-yellow fluorescence is very
striking and has been suggested as a test for the colon bacillus. Many other organ-
isms, especially those of the hog cholera group, have this power. It is convenient,
however, to color glucose bouillon with about i% of a %% solution of neutral red.

On the plates made for the detection of colon bacillus may be found
certain organisms which have origin in fecal contamination. The more
important of these are those of the paratyphoid, cloaca and proteus
groups. In addition, the B. fecalis alkdigines has not rarely been
isolated. Among natural water bacteria there may be present either
the liquefying or the nonliquefying B. fluorescens. These colonies
have a yellowish-green fluorescence.

Certain chromogenic cocci and bacilli are found in uncontaminated waters as
B. indicus or B. violaceus. From surface washings we obtain certain soil bacteria as
B. mycoides, B. subtilis, B. megatherium. One of the higher bacteria which shows
long threads, Cladothrix dichotoma, is common, and is characterized by a brown halo
around its gelatin plate colony.

BACTERIOLOGY OF MILK 155

Isolation of the Typhoid Bacillus from Water

This is probably the most discouraging procedure which can be
taken up in a laboratory. Only the most recent reports of such isola-
tion from water supplies, which have been verified by immunity reac-
tions, can be accepted and of these the number of instances is exceed-
ingly small. Owing to the long period of incubation, the typhoid
organisms may have died out before the outbreak of an epidemic
suggests the examination of the water supply.

There have been various methods proposed for the detection of the B. typhosus in
water. A method which would offer about as reasonable a chance of success as any
other would be to pass 2 or 3 liters of the water through a Berkefeld filter; then to
take up in a small quantity of water all the bacteria held back by the filter. Then
plate out on lactose litmus agar and examine colonies which do not show any pink
coloration. The dysentery bacillus has about the same cultural characteristics as
the typhoid one, so that it is important to note motility. If from such a colony
you obtain an organism giving the cultural characteristics of B. typhosus, carry out
agglutination and preferably bacteriolytic tests as well. Some strains of typhoid,
especially when recently isolated from the body, do i\ot show agglutination.

The Conradi Drigalski, the malachite-green, and various caffeine containing plat-
ing media have been highly recommended.

Isolation of the Cholera Spirillum from Water

The method proposed by Koch in 1893 does not seem to have been improved upon
by later investigators. To 100 c.c. of the suspected water add i% of peptone
and i % of salt. Incubate at 38°C., and at intervals of eight, twelve, and eighteen
hours examine microscopically loopfuls taken from the surface of the liquid in the
flask. So soon as comma-shape organisms are observed, plate out on agar. The
colonies showing morphologically characteristic organisms should be tested as to
agglutination and bacteriolysis. Inasmuch as the true cholera spirillum shows a
marked cholera-red reaction it is well to inoculate a tube of peptone solution from
such a colony and add a drop of concentrated sulphuric acid after incubating for
eighteen hours. The rose-pink coloration is given by the cholera spirillum with the
acid alone— the nitroso factor in the reaction being produced by the organism.

BACTERIOLOGICAL EXAMINATION OF MILK

A bacterial milk count is of comparatively little value as showing
whether a milk is dangerous or not. As a matter of fact, a milk which
contains several million of bacteria per c.c. might be less dangerous
than one containing only a few thousand, especially if in the latter
there were numerous liquefiers and gas producers present. There Js,

156 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

however, one point of importance in connection with the quantitative
estimation of bacteria in milk, and that is the fact that in order to keep
the development of the bacteria within the limits of 10,000 to 50,000
per c.c., it is necessary that the requirements of cleanliness in milking
and the rapid cooling of the milk after obtaining it and the keeping of
the temperature below 5o°C. be rigidly observed. If a milk has a
high count it shows some error in the handling of the milk. Anderson
has found that top milk contains from 10 to 500 times as many bacteria
as bottom milk. Centrifugally raised cream contains more bacteria
than that forming by gravity. In making a quantitative bacteriolog-
ical examination, the principle is the same as with water.

Make a known dilution of the milk with sterile water; add definite quantities of
this diluted milk to tubes of melted agar or gelatin, and pour into plates. The
diluted milk may also be delivered in the center of the plate and the melted agar or
gelatin poured directly on it, mixing thoroughly. Always shake the bottle well
before taking sample.

Example. — Added i c.c. of milk to 199 c.c. of sterile water in a large flask (500 to
looo c.c.). After shaking thoroughly, take i c.c. of this i : 200 dilution and add it to
99 c.c. of sterile water. Shaking thoroughly, we have a dilution of i : 20,000. Of this
we added 0.5 c.c. to a tube of gelatin or agar. After incubation the plate showed
75 colonies. Therefore the milk continued in each c.c. 75 X 2 X 20,000 (dilution)
= 3,000,000 — the number of bacteria in each c.c. of milk.

Lactose litmus gelatin or agar is to be preferred in milk-work, as the normal lactic
acid bacteria produce reddish colonies which are very striking. A standard easily
attained for high-grade, certified milk would be 5000 to 10,000 per c.c.

In the qualitative examination of milk, many dairies employ the fermentation
tube, any organism producing gas being considered undesirable. Again liquefying
organisms, as shown by the presence of such bacteria in the gelatin plates, are evidence
of probable contamination by faecal bacteria. A question which seems difficult to
decide is as to the general nature of the so-called normal lactic acid bacteria of milk.
Some describe them as very short, broad bacilli with very small colonies, fermenting
lactose with the formation of lactic acid. Others consider that the streptococci are
the organisms which are concerned with the normal fermentative changes. In
examining specimens of milk considered the best on the market, 1 have repeatedly
found the small red colonies on lactose litmus agar to be in chains of either Gram-
positive streptococci or streptobacilli.

Acid Producing Organisms. — Shippen considers the chief organism
concerned in the souring of milk as B. giintherii, but notes that it is
the same organism as S. lacticus. All authors note the difficulty of
deciding whether the morphology is coccal or bacillary. McGuire
found these organisms almost constantly present in the dung of cows.
The organisms he obtained from cow dung were chiefly members of
the coli-aerogenes group and S. lacticus.

BACILLUS BULGARICUS 157

Of the acid-forming bacilli in milk we have i. the B. lactls acid group. These are
oval cells about 0.9 microns by 0.6 microns, often in chains. They are Gram-
positive and nonmotile. They may be the same as Streptococcus lacticus of Kruse.
They curdle milk with a homogeneous clot — this being due to the fact that they do
not produce gas in lactose media. 2. The B. coli aerc genes group. These are gas
producers. (See under water.) 3. The B. bulgaricus group. In connection with
the organisms present in the tablets used for treating milk to produce lactic acid for
the treatment of intestinal disorders, and considered to be normal lactic acid bacteria,
I have found both streptococci and bacilli. These have all agreed, however, in not
producing gas in either lactose or glucose fermentation tubes.

I have often found the commercial fluid cultures sterile, the great
acidity produced by B. bulgaricus causing this. Fresh tubes may show
an acidity of +12 or about 10 times that of ordinary culture media.

The organism upon which special stress is laid in these so-called lactic acid pro-
ducers is the B. bulgaricus. This is a large, nonmotile organism with square ends
like anthrax. It often occurs in long chains and does not possess spores. It is Gram-
positive and often shows metachromatic granules like those of the diphtheria bacillus.
Colonies show in forty-eight hours which resemble streptococcus ones, but are more
contoured on the surface. Magnified the colonies resemble young mould colonies
It grows better on milk agar plates than on whey agar plates. The opacity of the
milk agar plate is but a slight objection. It produces a deep vivid pink in litmus
milk, while milk streptococci only cause a light pink. It produces a very large
amount of acid (3%). Little or no growth on ordinary laboratory media or bejow
o°C. (Op. temp. 42°C.).

Heinemann states that it occurs normally in human faeces and various fermented
milks — also in gastric juice when HC1 is absent. To isolate, put milk or faeces into
a broth containing 0.5% acetic acid and 2% glucose. Transfer to litmus milk after
twenty-four hours and from such tubes plate out on milk serum agar (coagulate
boiling milk with a few drops of acetic acid, filter and add i% peptone, 2% glucose
and 1.5% agar).

As they grow in very acid media the term acidophilous is applied. It was sup-
posed that these bacteria were peculiar to certain fermented milks as matzoon and
yogurt. Hastings has shown the group to be present in milk in the United States
and considers the source to be the alimentary tract of cows.

Milk Leukocytes. — Another source of information as to the quality
of a milk may be derived from a study of the number of leukocytes or
pus cells contained in i c.c. of the milk. It must be understood that
cellular elements which differ only slightly from true pus cells may be
found in the milk of healthy cows and may be found in great numbers.
Statements have been made that such cells are neither amoeboid nor
phagocytic.

The Doane-Buckley method is probably the most accurate. In this you throw
down the cellular contents of 10 c.c. of milk in a centrifuge revolving about 1000

158 BACTERIOLIGY OF WATER, AIR, MILK, ETC.

times a minute for ten to twenty minutes. Then remove supernatant milk and add
0.5 c.c. of Toisson's solution to the sediment. Instead of Toisson's solution I use
Gram's iodine solution which brings out the leukocytes equally well and gives a cleaner
preparation. You thus have the leukocytes of 10 c.c. contained in 0.5 c.c. (Con-
centrated 20 times.) Make a haematocytometer preparation as for blood and
find the average number of cells for each square millimeter. Then multiply this by
10 to get the number of cells in a cubic millimeter. As a cubic millimeter is 1000
times smaller than a cubic centimeter, you multiply the number per cubic milli-
meter by 1000. Then, as the milk was concentrated 20 times, you divide by 20.
(If it were diluted 20 times, you would multiply by 20.)

Example. — Found an average of 50 cells per square millimeter. This would make
500 per cubic millimeter, and 500,000 per c.c.; then 500,000 divided by 20 would
give 25,000.

There is no agreement as to a standard for allowable leukocytes. Even in ap-
parently healthy animals they may exceed 100,000 per c.c. Doane has suggested
500,000 per c.c. as a preferable limit.

The smear methods for determining the number of leukocytes present do not
compare in accuracy with the volumetric ones. It is important, however, from a
standpoint of examining for tubercle bacilli, etc., as well as for recognition of leuko-
cytes, to deposit the sediment from a centrifuge tube, taken up with a capillary bulb
pipette, on a glass slide. Smear out slightly and then when dry fix with a mixture of
ether and absolute alcohol. Flood with ether to get rid of remaining fat and stain by
Gram's method or by acid-fast staining.

To summarize, we may state that the bacterial count is an indicator
of the care used in handling the milk while the presence of harmful
bacteria (qualitative examination) or numerous pus cells indicates
disease in the cow. During 1912 severe epidemics of sore throat due
to a streptococcus, S. epidemicus, were traced to milk of cows having
probably suffered from mastitis. In Baltimore the milk had been
pasteurized by the flash method which indicates the unreliability of
this process.

Pasteurization of Milk. — The objections to this method of preserving milk
have been (i) that the lactic acid bacteria which have been by some credited with
antagonism to harmful bacteria, would be destroyed by pasteurization, (2) the more
rapid development of bacteria in milk that has been pasteurized (3) interference with
nutritive qualities and (4) pasteurized milk does not show its deterioration as does
unpasteurized milk, thus failing to give a clue as to the age of the milk.

The United States Bureau of Animal Industry in studying this important phase of
the milk question has grouped the milk bacteria into three classes (a) acid-forming,
(b) putrefactive (liquefying) and (c) inert bacteria. In their investigations it was
found that many acid-forming bacteria withstood temperature as high as i68°F.,
so that pasteurized milk was soured just as is raw milk, but more slowly. They found
that pasteurized milk showed fewer putrefactive bacteria than raw milk, so that
even should it be a fact that injurious toxins were produced by spore-bearing putre-

BACTERIOLOGY OF AIR

159

factive organisms the development of such organisms would be even less in pasteur-
ized milk.

The statement so often advanced that bacteria develop more rapidly in pasteurized
milk than in raw milk was proved fallacious.

It was recommended that holding the milk for thirty minutes at i45°F. was a far
better method of pasteurizing than quickly bringing the milk to a temperature of
i85°F. (flash method). All admit the great value of the killing of important patho-
gens (typhoid, cholera, streptococci, etc.).

BACTERIOLOGICAL EXAMINATION or AIR

In Paris a cubic meter of air was found to contain the following
number of organisms:

Suburbs. — Winter,

Summer,

City Hall.— Winter,
Summer,

145 moulds,

245 moulds,

1345 moulds,

2500 moulds,

170 bacteria.

345 bacteria.
4305 bacteria.
9845 bacteria.

Air of hospitals, especially after sweeping, may contain 50,000
bacteria per cubic meter. There does not seem to be any particular
relation between the amount of carbon dioxide in air and the bacterial
content.

Petri's Rough Method. — Exposure of a lactose litmus agar plate (capacity 100
sq. cm.) for five minutes will give the number of organisms present in 10 liters of air.
Multiply by 100 for i^cu. m.

FIG. 44. — Sedgwick -Tucker aerobiscope. (Mac Neat.)

The two groups of organisms usually found in air are i. bacteria and 2. moulds.
Moulds (spores) may be carried by currents of air; bacteria, however, are generally
carried about by particles of dust or finely divided liquids (spray). On the lactose
litmus agar plate staphylococci and streptococci show as bright red colonies.

Sedgwick-Tucker Sterile Granulated Sugar Method. — Sterilize aerobioscope
and introduce granulated sugar on support. Again sterilize (not over i2o°C. in
dry-air sterilizer). Allow a given quantity of air to pass through; then shake the
sugar into wide part of aerobioscope. Now pour in 10 or 15 c.c. of melted gelatin
(40° C.) to dissolve sugar. Roll tubes as for Esmarch roll cultures, and incubate
at room temperature. To draw air through the aerobioscope, connect the small end
with a piece of rubber tubing which is attached to a tube in the stopper of an aspirat-
ing bottle. Having poured a definite quantity of water into the aspirating bottle,

160 BACTERIOLOGY OF WATER, AIR, MILK, ETC.

allow the water to run out. The same quantity of air will be drawn through the
sugar of the aerobioscope as the amount of water passing out of the aspirating bottle.
The bacteria and moulds are caught by the sugar.

Example. — Passed 10 liters of air through the aerobioscope. The bacteria in this
quantity of air showed 75 colonies when incubated at 2O°C. The unit being i
cu. m. or 1000 liters, we have only obtained the bacteria of one hundredth of the
unit. Hence multiplying 75 by 100 gives 7500 bacteria as present in i cu. m. of
the air examined.

A very satisfactory method is to take a test-tube containing 5 c.c.
of sterile water and having a rubber stopper with two perforations,
one for a long piece of glass tubing which dips down into the water
and a short piece of glass tubing which is connected with the aspirating
bottle by rubber tubing.

The air to be examined is drawn through the long tube and its bacterial or mould
content is caught in the water. By plating i c.c. which would represent one-fifth
of the total count for the amount of air aspirated we can easily calculate the con-
tent for a cubic meter.

In comparing the results with the aerobiscope with those obtainec
by exposing a plate as in Petri's method for ten instead of five minutes,
it was found that the latter was sufficiently in accord to make it a satis-
factory approximate quantitative method. The simplicity and ease of
access to the colonies developing on it make it preferable when the air of
operating-rooms or hospital wards is to be examined.

Of the fungi ordinarily obtained in examinations of the air the blue-green mould
and 'the red yeast are the most common. B. subtilis and sarcina types of cocci are
the most common bacterial colonies found upon exposed plates. Sewer air is as a
rule free from bacteria, due probably to the fact that bacteria tend to adhere to
moist surfaces. The importance of Fliigge's droplet method of contamination of
the air of a room is brought out in the discussion of infection with pneumonic plague.
This is an important method in the transmission of tuberculosis.

CHAPTER XII
PRACTICAL METHODS IN IMMUNITY

THAT which prevents the gaining of a foothold by disease organisms
in the animal body or which neutralizes their harmful products or de-
stroys the parasites is termed immunity. In the main, the question
of immunity hinges on the powers of resistance of the human body
and the aggressiveness or virulence of the invading organism. It must
always be kept in mind that immunity is only relative; thus the fowl,
which is practically immune to tetanus, may be made to succumb by
reducing its resistance by refrigeration or by increasing the amount of
poison introduced. The insusceptibility which the fowl has to tetanus
or which man has to many diseases of animals is best termed inherent
or inborn immunity, and is at present only a subject of theoretical
interest. When immunity to a given disease is obtained as a result of
an attack of the disease in question or bvJahoratory methods of inocu-
lation, this is termed properly an acquired immunity, and injthe former
case is anna.t.ii rally acquired immunity and in the second an artificially
acquired immunity.

Immunity then may be divided into that which is inherent and that which
is acquired. Inherent immunity is such as is observed in the resistance of Algerian
sheep to anthrax (ordinary sheep are very susceptible) or the fowl to tetanus and
is of interest theoretically rather than practically. Acquired immunity may be
brought about naturally as by an attack of a disease or artificially by laboratory
measures.

As a result of an attack of a disease, which may be regarded as
accidentally acquired, or in response to the stimulus of the injection of
the organism or its products, we have developed in the man so infected
or injected certain specific antagonistic properties to that organism,
which are usually demonstrable in the blood-serum or other body fluids,
and to which we apply the terms agglutinating power, precipitating
power, opsom'c power, or bacteriolytic power. The term antibody is
also applied. All four powers may be present together in equal or in
varying degree or one or more may be absent. By agglutinating power
we mean that which causes evenly distributed organisms to come
ii 161

162

PRACTICAL METHODS IN IMMUNITY

together and form clumps. By precipitating power we mean the
ability of such a serum to cause precipitates in a clear bouillon filtrate
of the specific bacterium. Such antibodies are called precipitins or
coagulins. Bfv^/psonic power we mean that antibody which so alters
tne resistance of bacteria that the phagocytes ingest them. By^pac-
teriolytic poj^er we mean that which brings about disintegration or lysis
of the specific organism. The bacterium which causes the disease or
which is used in inoculation for the production of immunity is termed
the specific organism.

FIG. 45. — Receptors of the first order uniting" with toxin. (Journal of the Ameri-
can Medical Association, 1905, p. 955.) a, Cell receptor; b, toxin molecule;^ c,
haptophore of the toxin molecule; d, toxophore of the toxin molecule; e, haptophore
of the cell receptor.

Artificially Acquired Immunity. — Of the different kinds of immunity only artificial
immunity will be considered. This may be obtained in two ways : i. By injecting
the bacteria or their products into man or animals and as the result of the activity of
the cells of the animal invaded, antibodies' are formed which neutralize the toxins
(anj^tp^is) or bring about lysis W Ine specific bacteria (baclmojysins). These
antibodies which are supposed to be thrown off (free receptors) from those body cells
which have suitable fixation powers for the invading toxin molecule or bacterium
may remain potential for months or years and so confer a more or less enduring
immunity.

These fixation points are known as cell receptors and are intended for
the assimilation of various foodstuffs by the cell. If destroyed by the
toxin or bacterium they are reproduced in great excess by nature.

ACTIVE AND PASSIVE IMMUNITY

I63

Not only may bacteria act in this way but foreign cells, such as red cells or various
parenchymatous cells, when injected, give^rise to antagnnisHr substances which act
as factors in their destruction— haemolysins for red cells, cytolysins jor different
parenchymatous cells. Such methods produce " active immunity."

The substance which is injected and in reaction to which antibodies
are produced is called a,n]antigen.

2. When we take the serum of a man or animal immunized actively and inject
it with its contained antibodies into a second animal or man, we confer an immunity
on the second animal; but as his cells take no active part in the production of the
immunity, but are only passive, we term this immunity "passive immunity." If
this serum which is introduced in passive immunity only neutralizes the toxic prod-

FIG. 46. — Receptors of the second order and of some substance uniting with one
of them. (Journal of the American Medical Association, 1905, p. 1131.) c, Cell
receptor of the second order; d, toxophore or zymophore group of the receptor; e,
haptophore of the receptor; /, food substance or product of bacterial disintegration
uniting with the haptophore of the cell receptor.

ucts of the infecting bacteria, we term it antitoxic passive immunity and designate
the immune serum as antitoxic serum. If it destroys the organism, we call it_anti-
microbic serum, and the immunity, antimicrobic passive immunity. Some immune
sera are both antitoxic and antimicrobic.

Toxins. — It is well to remember that some organisms produce ajsoluble or^extra-
cellular toxin which is given off while the bacterium is alive: and in other instances
the toxin is Jntracellular and is only given off when the bacterium disintegrates:
consequently, an antimicrobic serum may cause the liberation of toxin. Diphtheria,
tetanus, or b^tujjism antisera are instances of antitoxic sera, while practically all others
a7e""antimicrobic. The antidysentery serum against Shiga strains seems to have
antitoxic power. B. pyocyaneus also has a soluble toxin. There is but one factor to

164

PRACTICAL METHODS IN IMMUNITY

consider in an antitoxic serum and that is the protoplasmic particles which are
thrown off from the cell in response to the injury incident to the attack upon the cell
by the toxin particles. This free particle in the circulation represents the entire
mechanism of antitoxic immunity. It is capable of uniting with the toxin molecule
and neutralizing its toxic power, or rather so binding its combining end (naptophore
group) that it is incapable of attaching itself to a cell, so that the poisonous end of the
toxin (toxophore group) cannot have access to the cell.

The term toxin, strictly speaking, is applicable only to such bacterial
poisons as (i) require a period of incubation before being capable of
manifesting toxic symptoms and (2) can produce antitoxins.

For further discussion of toxins and antitoxins see under diphtheria, tetanus,
botulism, and pyocyaneus infections.

FIG. 47. — Receptor of third order, and of some substance uniting with one of
them. (Journal of the American Medical Association, 1905, p. 1369.) c, Cell recep-
tor of the third 'order — an amboceptor; e, one of the haptophores of the amboceptor,
with which some food substance or product of bacterial disintegration (/) may unite;
g, the other haptophore of the amboceptor with which complement may unite;
k, complement; A, the haptophore; z, the zymotoxic group of complements.

Antimicrobic Sera. — In antimicrobic sera we have two factors to
consider, the first is a prQt.np1f».smfr pajtirlp quite similar to the anti-
toxin molecule, but which in itself has no power of injuring its specific
bacterium. This particle is generally referred to as the amboceptor or
immune body. It is the specific product of the activity of a specific
bacterium or foreign cell against the body cells attacked. It with-
stands a temperature above q6°C. and of itself is incapable of injuring
the bacterium in response to whose attack it was produced. The
second factor in the bacteriolysis of the specific bacterium, or the haemoly-

COMPLEMENT 165

sis of the specific foreign cell, is something normally present in the
serum of every animal, and which is capable of disintegrating a foreign
cell or bacterium, provided it can have access to the cell or bacte-
rium through an intermediary ambpceptor (hence the amboceptor is
sometimes called an intermediary body). This something is called
the complement. It is by some called alexine, by others cytase
(Metchnikoff). ' ^_ _^^^^"

The complement cannot act upon and destroy an invading bacterium or cell unless
the amboceptor is present to make the necessary connection. The complement is
destroyed by a temperature of s6°C., so that, if we heat the serum from an immune
animal to s6°C., the complement it naturally contains is destroyed, and the ambo-
ceptor it contains, which is not injured by such a temperature, is incapable of de-
stroying bacteria or cells, unless we replace the complement which has been destroyed
by fresh complement. This is done experimentally by adding the serum of a non-
immunized animal which contains the complement, but no specifier immune body
(amboceptor), to the heated serum. This is termed "activating," and a serum so
treated is said to be "activated." When an immune serum has been heated to 56°C.,
it is said to have been "inactivated."

Antirnicrobic sera are not as efficient in treatment as antitoxic ones.
It might be that if we could use homologous sera for treating man instead
of the usual heterologous ones from the horse better results might
obtain.

It would appear that a more hopeful outlook will obtain by combining serum
therapy with chemo-therapy, thus a combination of antipneumococcic serum with
sodium oleate seems capable of producing curative results which neither alone can
bring about.

Again, a combination of vaccination (active immunization) with the injection of
the antimicrobic serum (passive immunization) has been thought by some to be of
value.

When we allow a mixture of bacteria or cells to remain in contact
with their specific imrrmne^serum which has been inactivated, the ambo-
ceptors attach themselves to the bacteria or cells, so tnat now, upon
adding normal serum (complement), these bacteria or cells are so
prepared or mordanted that the complement can disintegrate them.
This experiment of attaching amboceptors to cells is termed sensitizing
and cells so treated are said to be sensitized.

METHODS FOR OBTAINING IMMUNE SERA

While a convalescent from a disease may be utilized to obtain an
antitoxic, agglutinating, opsonic, or bacteriolytic serum against the

i66

PRACTICAL METHODS IN IMMUNITY

specific bacterium, yet this is more conveniently obtained from an
animal which has been immunized against the bacterium or cell in
question. The rabbit is the most convenient animal to employ for the
production of immune sera where the object is to have at hand a serum
for use in diagnosis.

Where sera are used on an extensive scale, as in the production of
curative sera, larger animals are employed. There are two applications
of serum diagnosis: i. Where the bacterium is known and the serum
is to be diagnosed. 2. Where the serum is known and the bacterium
is to be diagnosed.

The first is employed by testing the agglutinating or bacteriolytic power of the
serum taken from a patient upon pure cultures of the organism which is suspected as
the cause of the disease. The Widal test (agglutination) is the best instance of this
procedure. This method is of practical value in the diagnosis only of typhoid, Malta
fever, and para-typhoid. In diseases like cholera and bacillary dysentery, the
disease has run its course before agglutinating power becomes apparent in the
serum. This method, however, may be used to prove that a convalescent has suf-
fered from a suspected disease. Thus, by testing the agglutinating power of a
serum, one or two weeks after recovery from a suspicious case of ptomaine poisoning,
we may be able to demonstrate that the case in question was cholera. The second
method has wider application, and is the one in which we use the sera of animals
which have been immunized with known bacteria. Organisms isolated from urine,
faeces, or blood of patients, or those obtained from water or food supplies may be
identified by testing the agglutinating, opsonic, or bacteriolytic power of known
sera against them. This has a wide range of applicability. The testing of the
opsonic power of the sera in man or animals immunized against plague, and possibly
cerebrospinal meningitis, seems to give more definite information than do agglutina-
tion or bacteriolytic tests. With the majority of other organisms, however, the
agglutination test is the one almost always preferred.

Even in a small laboratory there are no particular difficulties in the way of having
on hand rabbits immunized against typhoid, paratyphoid, Malta fever, acid-
producing and nonacid-producing strains of dysentery, cholera, etc. Just as we
inject men with vaccines prepared from various bacteria in opsonic therapy, so we
inject animals to produce sera for diagnosis. We may use either a bouillon culture
or the growth on agar slants taken up with salt solution as the inoculating material.
This is heated for one hour at 6o°C. to kill the bacteria. Where we desire to produce
a serum which will disintegrate red blood cells (hiemolytic serum), we inject intra-
venously about i c.c. or intraperitoneally about 5 c.c. of the washed red cells of the
animal for which we wish to produce a specific serum. For details see method of
preparing haemolytic amboceptor serum under Noguchi's modification of Wassermann
test.

Precipitating Sera. — For preparing a serum for the biological blood test we inject
the rabbit intravenously with human serum in quantities of about 5 c.c. every fifth
day. About one week after the last injection the antiserum obtained from the in-
jected rabbit should be strong enough for Ho c.c. to produce turbidity when added

AGGLUTINATING SERA

l67

to i c.c. of a i-iooo dilution of human serum in salt solution. Various controls are
necessary when used in medico-legal work.

Agglutinating Sera. — For obtaining an agglutinating or bacteriolytic serum for
bacteria we inject about i c.c. of the killed bacterial bouillon culture subcutaneously
or into the peritoneal cavity of the rabbit. The easiest way to inject the rabbit is to
hold the animal head down and plunge the needle in the median line into the ab-
dominal cavity, forcing in the contents of the syringe. The intestines gravitate down-
ward and by entering the needle below the limits of the bladder we avoid injuring
any vital part. It may be more satisfactory to at first inject only about % c.c., and

FIG. 48.— i, Red cells + normal serum. No amboceptor. No haemolysis. A,
Complement; B, normal red cell. 2, Red cells 4-immune 'serum. Complement and
amboceptor. Haemolysis. C, Complement; D, amboceptor; E, haemolyzed red
cell. 3, Red cells + immune serum heated to 56.0C. Inactivated. Complement
destroyed. No haemolysis. F, Destroyed complement; G, amboceptor; H, red
cells. 4, Red cells + heated immune serum + fresh serum. (Activated by con-
tained complement.) Haemolysis. I, Destroyed complement; J, fresh complement;
K, amboceptor; L, heemolysed red cell. 5, Diagram showing antitoxin production.
a, Toxin molecule; b, antitoxin molecule; c, neutralization of toxin by antitoxin.
6, Diagram showing bacteriolysin. d, Complement; e, amboceptor; /, bacillus.

then if there is very little reaction, as shown by the appetite and spirits of the rabbit,
to inject about four days later i c.c. About four or five injections at intervals of
three to five days will usually produce an immune serum.

Injection of the antigenic material (blood cells, serum or bacterial emulsion) into
the marginal ear vein may be employed. With this method, however, I have had
several rabbits die in what was considered anaphylactic shock. (For the method
of immunizing rabbits to produce a haemolytic serum see Wassermann test.) Some

i68

PRACTICAL METHODS IN IMMUNITY

animals do not seem to be capable of producing antibodies, so that it may be neces-
sary to use one or more rabbits before a satisfactory serum is obtained. The most
convenient way of obtaining serum for a test is to cut across one of the marginal
veins of the rabbit's ear, and collect the blood in a Wright's U-tube. Centrifugaliz-
ing, we have the serum ready for use.

The vein can be made to stand out prominently by applying a compress dipped
into very hot water. When a large amount of serum is desired it is better to use a
test-tube with two pieces of glass tubing passing through a double perforated rub-
ber stopper. To one of the projecting pieces of glass tubing a stout hypodermic
needle is attached through the medium of 8 inches of rubber tubing and to the
second piece of glass tubing passing through the stopper of the large test-tube
another piece of rubber tubing is attached for suction. To obtain blood from the
rabbit find the ensiform cartilage and insert the needle in the notch to the left and
gently force it upward. Applying suction with the mouth the blood flows into the
test-tube as soon as the needle enters the heart. By placing the tube of blood in
the refrigerator the serum separates out from the clot. The removal of 20 to 30 c.c.
of blood does not seem to affect the animals in the least and they can be used in this
way time and time again. The immune body and agglutinin in serum remain
active for weeks when kept in the refrigerator. Such sera may also be dried on paper
as for amboceptor paper (Noguchi). The complement and opsonin, however, begin
to deteriorate at once and have disappeared by the fifth day. Consequently, for
opsonic and bacteriolytic and haemolytic experiments, fresh serum — twelve to twenty-
four hours — must be used, or it may be activated.

AGGLUTINATION TESTS

There are two methods of testing the agglutinating power of a
serum — the microscopical and the macroscopical or sedimentation
method.

The Widal Reaction. — i. For the microscopical method draw up serum to the
mark 0.5 of the white pipette. Then draw up salt solution to the mark n. This
when mixed gives a dilution of i to 20. (It is more convenient to make the serum
dilutions with a graduated rubber bulb capillary pipette.) One loopful of the diluted
serujn and one loopful of a bouillon culture or salt solution suspension of the organism
to be testeo" gives a dilution of i to 40. One loopful of the i to 20 diluted serum and
3 loopfuls of the bacterial suspension give a dilution of i to 80. These two dilutions
answer in ordinary diagnostic tests. The red pipette with a i to 100 to i to 200
dilution may be used where dilutions approaching i to 1000 are desired. Having
mixed the diluted serum and the bacterial suspension on a cover-glass, we invert it
over a vaselined concave slide and examine with a high power, a dry objective (}/§
i nch) . It is neater to press down the vaselined periphery of the concavity on the cover-
glass. This sticks to the borders of the cover-glass and the preparation is easily
handled. It is simpler to make a ring of vaseline to fit the cover-glass and make
the mixture of diluted serum and culture in the center of this ring or square. Then
apply the cover-glass, press it down on the vaseline ring and examine as with the
ordinary hanging drop. In making dilutions it is preferable to use salt solution, as

AGGLUTINATION 169

the phenomenon of agglutination requires the presence of salts. Ordinarily, thirty
minutes is a sufficient time to wait before reporting the absence of agglutination.
Agglutination is more rapid at body temperature than at room temperature. In
reporting agglutination, always give time and dilution. It is absolutely necessary
that a control preparation be prepared in every instance; that is, one with the
bacterial culture alone or with a normal serum of the same dilution as the lowest used.
Some normal sera will agglutinate in i to 10 dilution, and group agglutinations (as
paratyphoid with typhoid serum) may occur in i to 40 or possibly higher. It is very
unusual for sera to agglutinate any other bacteria than the specific one in dilutions as
high as i to 80.

Macroscopic Agglutination. — 2. For the macroscopical or sedimentation test, take a
series of small test-tubes (% X 3 inches) and deposit i c.c. of salt solution in each of
the series. Now, having taken an empty test-tube, drop 4 drops of serum in it and
then add 1 2 drops of salt solution. This approximately gives i c.c. of a i to 4 dilution
of the serum. It is more exact to make the i to 4 dilution with a graduated pipette.
With a rubber-bulb capillary pipette, which has been graduated to hold 16 drops or
i c.c., draw up the contents of the tube containing the i to 4 serum and add it to the
next tube containing i c.c. of salt solution. This gives 2 c.c. of a dilution of i to 8.
Now mix thoroughly by drawing up and forcing out with the bulb pipette, and then
withdraw i c.c. and add to the next tube containing i c.c. of salt solution. This
gives a dilution of i to 16. Having mixed as before, again withdraw i c.c. of the
mixture and add it to the i c.c. in the next tube. We now have a dilution of i to 32.
Again withdrawing i c.c. and adding it to the fourth tube containing i c.c. of salt
solution we have a dilution of i to 64. In tube i there is now i c.c. of a dilution of
the serum of i to 8; in tube 2, there is i c.c. of a dilution of i to 16; in tube 3 of i to
32. Tube 4 contains 2 c.c. of i to 64. Now adding i c.c. of a culture of typhoid
or any other organism, we have the dilution of the serum in each tube doubled.
Tube i now contains a serum in dilution of i to 16, acting on the bacteria; tube 2 of
a i to 32; tube 3 of a i to 64. Now place these tubes in the incubator and, after two
to five hours or overnight, we examine for the clearing up of the supernatant fluid.
If the serum in a certain dilution agglutinates, the clumps gravitate to the bottom
and the upper part becomes clear. If so desired, these dilutions may be carried on
to i to several hundred in the same way. It is safer to work with dead cultures in-
stead of living ones. To prepare, take a twenty-four-hour agar slant culture of
typhoid or paratyphoid and emulsify in salt solution (about 6 c.c. to a slant).

By adding o.i of i% of formalin to the typhoid emulsion and placing in the ice-
box the cultures will be found sterile in about three days. The emulsion should be
shaken twice daily while undergoing sterilization in the ice-box. Such cultures
are not easily contaminated and appear to retain their agglutinable qualities for
several months. The macroscopic methods are preferable with such dead cultures.

A very convenient method in general use in Germany is the follow-
ing: Make dilutions of serum in ordinary test-tubes (% X 6 inches)
as described for the small test-tubes.

Then take a loopful (2 mg.) of culture from an eighteen to twenty-four-hour-old
agar culture and emulsify it thoroughly in the dilution in the first test-tube—repeat
the process in the second tube and so on. This procedure is much safer than when

I70 PRACTICAL METHODS IN IMMUNITY

live cultures are added with a pipette. Again, the dilution is unchanged by this
addition whereas it is doubled when an equal volume of culture is added to the
diluted serum. A control should always be made in normal salt solution. After in-
cubating, observe flocculent precipitates (agglutination) by tilting the fluid in the
tubes to form a thin layer and to obtain the most advantageous light and look for a
fine curdy precipitate (agglutination) or a uniformly turbid emulsion (negative
reaction).

The method of using a slide with two vaselined rings, one containing
an emulsion in the specific serum and the other in salt solution is of
great practical value. This method is described under cholera.

Pfaundler under the designation of a thread reaction showed that organisms
tended to grow in thread forms in a culture medium containing the homologous
serum. Mandelbaum has suggested this as a means of diagnosing typhoid. Take
ordinary bouillon containing i % of sodium citrate. Inoculate it with a culture of
typhoid. Now with a bulb capillary pipette take up i part (as marked by a wax
pencil) of the patient's blood and 15 times as much of the citrated bouillon just
inoculated with typhoid. Mix the blood and citrated bouillon on a sterile slide or in
a test-tube and after drawing up into the lower part of the expansion of the capillary
pipette, seal off the capillary end. Now place the sealed-off pipette upright in an
incubator and after four or five hours take out from the expanded end a loopf ul of the
clear supernatant fluid (the blood cells settle to the bottom) and if the typhoid bacilli
are in chains instead of being single and motile it shows a positive reaction.

PRECIPITIN REACTIONS

In the diagnosis of bacterial infections the agglutinating tests are
so much more satisfactory that precipitin tests are rarely applied.
As will be noted under the Meningococcus such a test has been recom-
mended for the diagnosis of cerebrospinal fever.

In the technic of precipitin reactions for bacteria one filters two or three weeks
bouillon cultures of a given organism through a Berkefeld filter. (Precipitinogen.)
The filtrate should not only be perfectly transparent but also sterile as subsequent
bacterial growth would give turbidity similar to a positive reaction. The precipitin
containing serum is prepared by injecting rabbits intravenously with bacterial
filtrates as prepared above or with tne bacteria themselves. The methods are similar
to those for preparing agglutinating or haemolyzing sera.

Test: To four tubes each containing 2 c.c. of the bacterial filtrate
are added increasing quantities of the serum to be tested; 0.05 c.c. to the
first tube, o.i c.c. to the second and 0.5 c.c. to the third, and i c.c. to the
fourth. Controls of positive and negative precipitating sera should
also be prepared. The tubes are not shaken and the reaction should
be allowed five or six hours at room temperature before final readings

BIOLOGICAL BLOOD TEST 17 1

are made. When, the serum is strongly precipitating the clouding of
the clear fluid should take place in ten to twenty minutes.

In the biological blood lest rabbits are immunized intravenously either with whole
blood taken in citrated salt or with serum alone. For class work I use the blood of a
chicken injected intravenously into a rabbit. An immune serum thus prepared
contains haemolysins as well as precipitins. The haemolyzing effect of such a serum
on the nucleated fowl's red cells shows well when examined in a hanging drop. The
bleeding of the rabbit should be done after a period of fasting to avoid any opales-
cence of the serum.

Precipitating sera should be kept in the cold and may have one-tenth of i % car-
bolic acid added as a preservative, or they may be preserved on paper strips.

The suspected blood stain should be extracted with normal salt solution and
should be filtered until perfectly clear. An approximate strength of i to 1000 of the
blood is desirable. This can be estimated as given under albumin in urine, with the
U-shape tubing.

Test: Place 2 c.c. of the i to 1000 extract of the stain to be examined in tube i,
2 c.c. of i to 5000 in tube 2, and 2 c.c. of i«to 10,000 in tube 3, adding to each tube
o.i c.c. of the precipitating serum.

In another tube put 2 c.c. of a i to 1000 salt solution dilution of the serum of the
animal from which the suspected blood is supposed to come and add o.i c.c. of
precipitating serum.

Various other controls as with normal rabbit serum, etc., are necessary for medico-
legal application.

The tubes should not be shaken and may be kept at room temperature or in the
incubator. A positive reaction appears in two or three minutes as a clouding at the
bottom of the tube which becomes a distinct precipitate in fifteen or twenty minutes.
Readings should be made at the end of twenty minutes as reactions occurring sub-
sequently have no significance.

DEVIATION OF THE COMPLEMENT

It has been found that if there is not sufficient immune body in a
mixture of normal serum, containing abundant complement, and bac-
terial emulsion, only a portion of the bacteria will be destroyed. In-
creasing the amount of immune body with a constant quantity of normal
serum, we reach a point where all the bacteria are destroyed. Now,
if we continue to increase beyond this point the addition of immune
serum, the destruction of the bacteria ceases, and the cultures will again
contain myriads of living bacteria (Neisser-Wechsberg Phenomenon).

To carry out the test, make a series of tubes containing mixtures of bacteria with
the same quantity in each of normal serum. Thus, each tube contains % c.c. of
bacterial emulsion and ^ c.c. of i to 10 normal serum. Now inactivate a tube of
i to loo immune serum and to each of the tubes of normal serum and bacterial emulsion

172 PRACTICAL METHODS IN IMMUNITY

add increasing drops of the inactivated i to 100 immune serum. Thus, i drop to
No. i tube, 2 drops to No. 2 tube, and so on. After incubating for two hours, we
take a pipette and plate out a fraction of a drop in an agar plate. The limit at
which bacteriolysis is complete is shown by there being an absence of colonies.

Beyond or below that point colonies are more or less abundant. The explanation
of this phenomenon of deviation or deflection of the complement is that where we
have an excess of amboceptors for available receptors on the bacterial cells, only a
portion of the amboceptors can attach themselves to their specific bacteria. The
free amboceptors, not being able to form a union with the bacterial cell receptors
(for which they have a greater affinity), combine with the complement present.
Unless the complement be in excess, there will be no free complement left to join
on to the amboceptors attached to the bacterial cells, and consequently bacteriolysis
does not take place and the plate cultures show an abundance of colonies.

Stimson has found, in titrating his complement and amboceptor for complement-
fixation tests, that keeping his complement content constant and successively increas-
ing the amount of amboceptor gives increasing haemolyzing effect up to a certain
point, beyond which the further addition of amboceptor causes a lessening of
haemolytic power.

This he regards as due to deviation of complement and in his tests he prefers to
keep a fixed amount of amboceptor and adjust his titrations by increasing comple-
ment rather than amboceptor.

FIXATION OR ABSORPTION OF THE COMPLEMENT

One of the controversies in connection with the nature of the com-
plement is that regarding the question of the unity of complements
or whether there exist different kinds of complements for different
amboceptors (unity and multiplicity of complement). To prove that
a single complement will act with varying amboceptors, Bordet and
Gengou showed that the same complement would activate both haemo-
lytic and bacteriolytic immune bodies. If to a mixture of typhoid
bacteria and inactivated typhoid immune serum some guinea-pig serum
is added and the mixture allowed to remain at 37°C. for two hours,
and then sensitized red cells be added and the mixture again placed
in the incubator for two hours, no haemolysis will be found to have
occurred, because the bacteria have absorbed all the guinea-pig com-
plement through the intervening typhoid amboceptors, and there is no
complement left to haemolyze the red cells through the specific red
blood-cell amboceptors.

If, instead of immune typhoid serum, the serum of a normal person had been used,
there would have been no amboceptors to unite the complement to the bacterial
cells. The complement would then be at hand to unite with the sensitized red cells
subsequently added and bring about their haemolysis, as shown by the ruby red
color of the fluid.

REAGINE AND ANTIGEN 173

This phenomenon of Bordet and Gengou has been utilized by Wasser-
mann for the diagnosis of diseases where cultures are not applicable.
It is well recognized, however, that the body in a syphilitic serum
which reacts with the antigen is not an amboceptor but a lipoidophilic
substance, which has the property of linking complement to the
lipoidal antigen. The name reagine has been proposed for this lipoido-
philic substance. A similar substance is present in the serum of yaws
cases and the Wassermann reaction is just as constant in such cases as
in syphilis.

It is in the diagnosis of syphilis that it is best known. It having, until recently,
been impossible to obtain cultures of Treponema pallidum, we use an emulsion of the
liver of a syphilitic foetus, which has been filtered so as to be clear, instead of a
culture. The syphilitic liver, as can be observed by staining according to Levaditi's
method, is packed with spirochaetes.

Antigen. — While Noguchi has recently obtained pure cultures of the organism of
syphilis yet the antigen prepared from such cultures was not found as satisfactory
by Craig and Nichols as that from the liver of a syphilitic foetus, cases of syphilis
which showed strongly positive tests with ordinary antigen not giving a positive test
with the specific antigen.

It has now been found that lecithin or, preferably, emulsions of various normal
organs may be substituted as antigen for the syphilitic liver, the antigenic power
being due to lipoids. Aqueous extracts contain in addition to lipoids, substances which
render the antigen unstable — alcoholic extracts are more stable and contain less
anticomplement. The preparation of acetone insoluble antigen is described under
Noguchi's method and that of syphilitic liver under the Wassermann reaction.

Many prefer to use cholesterinized antigen. For its preparation:

Prepare guinea-pig, beef or human heart muscle as described under acetone in-
soluble antigen and extract 50 grams of this finely cut up muscle with 500 c.c.,
absolute alcohol for two weeks at 37°C. Then filter and add to one-half this
filtrate about 7 grams of C. P. cholesterin. Keep in 37°C. incubator over night and
then keep at a temperature of i6°C. for three hours. This precipitates excess of
cholesterin. Filter and to the filtrate add the other one-half of the heart extract,
giving an antigen half saturated.

In our laboratory we have found this antigen rather sensitive and
not altogether reliable and prefer to use the acetone insoluble one of
Noguchi.

For the immune bodies we take the serum of the patient, or if a case
of locomotor ataxia or general paresis, the cerebrospinal fluid.

In using cerebrospinal fluid it is customary to employ o.i c.c., 0.2
c.c. and i c.c. quantities instead of the amounts given for blood-serum
as directed in the tests to follow.

174 PRACTICAL METHODS IN IMMUNITY

It is usual to expect strong fixation with a paresis fluid in the smallest
amount noted above. For tabes use more fluid, as 0.5 and i c.c.

NOGUCHI'S METHOD

For the suspension of red cells use a J^% suspension of washed
human red cells.

For complement use fresh guinea-pig serum in a dilution of i part
to i>£ parts of salt solution (40%).

Method.— Take four small test-tubes (12 by 125 mm.) label la, ib and 20, 2&, re-
spectively. Into la and ib each put i drop of the serum of the patient to be tested
and into ia and 26 each put i equal size drop of the serum of a person known to give a
positive test for syphilis. The small drop delivered by a very finely drawn-out
capillary pipette, held vertically, is equal to about 0.02 c.c. or 50 drops to i c.c.
Next add to each of all four tubes i c.c. of the ^ % suspension of washed red cells.
Then add to each tube o.i c.c. of the 40% fresh guinea-pig serum. Now add
to tube ia and tube 2a each o.i c.c. of the i to 10 antigen dilution (opalescent working
antigen emulsion). Tubes ib and zb are controls not containing antigen. Mix con-
tents of tubes thoroughly and incubate at 37°C. for one hour or for one-half hour
in a water-bath. Now add to each of the four tubes 2 units of the immune haemolytic
serum, as measured off on the amboceptor paper strip — thus with a paper of which 2
mm. equals i unit, drop into each tube 4 mm. of the strip.

The tubes without antigen (ib and 2 b) should show good haemolysis.
Tube 2d, that of the known syphilitic, with antigen, should not show
haemolysis and that of the person examined (ia) should show haemolysis
in case the test is negative for syphilis. Moderately positive cases may
show a slight trace of haemolysis. Where the tubes without antigen
are without colour (no haemolysis) it shows that there is some anti-
complementary factor at work and that the tests should be regarded
as unsatisfactory.

This failure to haemolyze in the control tubes may be due to anticomplementary
substances in the serum or to the bacterial contamination of the serum, the proteids
of the disintegrating bacteria possibly preventing haemolysis by antigenic action and
thus absorbing the complement, which otherwise would bring about haemolysis.

It is advisable also to employ the serum of a person known to be
free from syphilis. In this case we should use two additional tubes,
30 and 36, conducting the test as for the syphilitic control serum.

If a normal serum is not used then use a system control in which there are put all the
reagents noted above except the human sera from the patient or the syphilitic
control.

When examining a large number of sera at the same time it is well to use rubber

NOGUCHI TEST 175

adhesive plaster labels with the name of the patient written on it as well as the number.
The greatest care should be exercised to avoid mistakes in numbers.

Many workers prefer to use inactivated serum for the test. In this case we should
add four times as much of the inactivated serum as for the unheated serum (0.08
instead of 0.02).

Inactivation not only destroys complement but likewise diminishes
the strength of the reagine content of the serum. Factors such as
character of food and general condition influence the complement
strength of guinea-pig serum so that it is advisable to titrate the guinea-
pig serum. To do this take i c.c. of a J^% emulsion of human red
cells and drop in i unit of amboceptor paper. The amount of com-
plement which will entirely haemolize the red cells in one-half an hour in
water-bath equals i unit of complement. For the test use 2 units of
complement.

Some workers prefer to use a single unit of complement and 2 units of ambo-
ceptor, thus doubling the amboceptor unit. Stimson has found, however, that
increasing amboceptor content may cause a deviation of Complement, so that he prefers
to use a single unit of amboceptor and 2 units of complement. In the original
Wassermann technic the units of both complement and amboceptor are doubled.
In the Noguchi only the amboceptor unit is doubled.

Preparation of Acetone Insoluble Antigen.— Take about 50 grams of finely divided
beef, dog, or rabbit heart or liver and triturate in a mortar to a paste. Pour on this
paste 500 c.c. of absolute alcohol and keep the mixture in a corked bottle in the 37°C.
incubator for five to seven days shaking the emulsion four or five times daily. (We
use beef heart and carefully pare away all fat and fibrous tissue and macerate the re-
maining muscular tissue with absolute or 96% alcohol. Next filter through paper
and collect the filtrate in a large shallow dish and hasten evaporation with the aid
of a current of air from an electric fan directed upon the surface. It is advisable to
cover the dish with a single layer of cheese cloth to prevent access of flies, etc., to the
contents. These insects may contaminate the solution with moulds which causes
disappearance of the lipoids.

Within twenty-four hours only a sticky residue should remain. This is taken up
in about 50 c.c. of ether and the turbid ethereal solution kept over night in the re-
frigerator in a corked bottle.

In the morning there will be found about 45 c.c. of clear supernatant fluid which is
decanted off and allowed to evaporate to about 15 c.c.

Now to this 15 c.c. add about 150 c.c. of acetone and a precipitate will form
which collects at the bottom of the measuring cylinder. Now pour off the supernatant
acetone and let the sediment stand until it is of a resinous consistence. Now dis-
solve 0.3 gram in i c.c. of ether and then add 9 c.c. of methyl alcohol. This gives the
stock antigen solution which is crystal clear.

In using this antigen solution for the Emery or Noguchi test we dilute i c.c. with
9 c.c. of salt solution. This opalescent, working, antigen emulsion should be made up
fresh on the day of preparing the tests.

176 . PRACTICAL METHODS IN IMMUNITY

About one-half of these antigens are lacking in power to absorb
complement in the presence of syphilitic sera. More rarely they may
absorb complement with a nonsyphilitic serum (anticomplementary)
or they may have a haemolytic action. Consequently a new stock
antigen should be tested as to its reliability —

1. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, and o.i
c.c. of a 10% suspension of washed red cells when incubated at 37°C. for two hours
should not show any haemolysis.

2. A mixture of 0.4 c.c. working antigen emulsion, 0.6 c.c. salt solution, o.i c.c.
of a 40% solution of fresh guinea-pig serum, and 2 units of amboceptor and incubated
at 37°C. for one hour should show haemolysis when we now add o.i c.c. of a 10%
washed red-cell emulsion and the whole then again incubated for two hours at 37°C.
(The antigen did not absorb complement in the absence of syphilitic antibodies.)

3. A mixture of 0.2 c.c. of a i to 10 dilution of working antigen emulsion, 0.8
c.c. of salt solution, i drop of syphilitic serum, o.i c.c. of a 40% dilution of fresh
guinea-pig serum, and 2 units of amboceptor should be incubated at 37°C. for one
hour. When we then add o.i c.c. of a 10% suspension of washed red cells and
again incubate for two hours we should fail to obtain haemolysis. (The antigen can
absorb complement through the intermediation of syphilitic antibodies.)

Preparation of Amboceptor Paper. — In order to secure blood from the vein of a
man or the heart of the immunized rabbit, the most convenient method is with the
use of an Erlenmeyer flask with a rubber stopper having two perforations in the
stopper. To one of the projecting pieces of glass tubing a stout hypodermic needle
is attached through the medium of about 8 inches of rubber tubing, and the second
piece of glass tubing is bent at an angle as it leaves the stopper to provide a suction
tube. With a man, constrict the upper arm sufficiently to stop venous return with
an Esmarck rubber bandage or a towel. Paint tincture of iodine over a prominent
vein at the bend of the elbow. Gentle suction will cause the blood to flow into the
needle tube and thence into the flask when the vein is entered.

The blood as it is taken from the arm should be received in about 50 c.c. of normal
salt solution containing i% of sodium citrate. About 28 to 30 c.c. are usually suffi-
cient. Now throw down this red-cell suspension in three or four centrifuge tubes.
The resulting sediment should be washed and rewashed with salt solution. Two
to three washings with salt solution suffice.

Now take a large healthy rabbit, shave the lower abdomen and paint the surface
with tincture of iodine. The easiest way to inject the rabbit is to hold the animal
head down and plunge the needle of a large glass hypodermic syringe containing the
washed red-cell sediment into the abdominal cavity in the median line. The intes-
tines gravitate downward and by entering the needle below the limits of the bladder
we avoid injuring any vital part.

Make the injections at intervals of five days and give increasing amounts at each
successive injection. Thus, first injection, 5 c.c.; second injection, 8 c.c.; third
injection, 10 c.c.; fourth injection 12 c.c.; and at the fifth injection give about 15 to
20 c.c. of washed red-cell sediment. It is well to dilute the cell sediment with an
equal amount of salt solution. About ten days after the last injection, we take some
blood in a Wright's tube from a vein of the ear and dilute the serum to make a i to

THE WASSERMANN TEST 177

100 dilution. To i c.c. of a J^% emulsion of red cells we add o.i c.c. of a 40% dilu-
tion of guinea-pig's fresh serum — similar combinations being made in a series of
10 tubes. To each of these tubes we add varying amounts of the i to 100 dilution,
o.i c.c. in the first, 0.2 c.c. in the second, 0.3 c.c. in the third, and so on. If we
obtain haemolysis in the tube containing 0.2 c.c. of i to 100 dilution of serum but not
in that containing o.i c.c. we note that the serum has a titer of about i to 500. If
the o.i c.c. gave haemolysis, the serum would have a titer of i to 1000.

Having ascertained that the haemolytic serum is sufficiently strong we shave the
left side of the thorax of the rabbit and enter the needle of the apparatus similar to
that used for taking the blood from a man's vein in one of the intercostal spaces of the
left side.

Having introduced the needle, feel for the heart beat and then plunge the needle
into the heart. We can withdraw about 30 c.c. of blood without injury to the rabbit.
This blood should be received in a clean empty flask and set, over night, in the re-
frigerator. The following morning pour off the clear serum into a clean Petri dish
and saturate, one by one, squares of filter paper with the serum. Allow the filter
paper to dry on a piece of unbleached muslin. Noguchi recommends Schleich and
Schull's paper No. 597. When thoroughly dry cut strips 5 mm. wide. This makes
the amboceptor paper. To standardize, take a series of tubes containing i c.c. of
a K% emulsion of red cells and add o.i c.c. of 40% dilution of guinea-pig serum for
complement. Next cut across the amboceptor paper strip pieces of varying width,
as i mm., 2 mm., 3 mm., 5 mm., and so on. The narrowest strip which gives haemo-
lysis in one hour in the incubator or one-half hour in the water-bath equals i unit.
Thus if a piece 5 mm. wide was required to produce haemolysis, 5 mm. of the paper
would have a value of i unit.

Coca injects rabbits intravenously with i c.c. of well-washed red cells
and five days later gives a similar dose intravenously. The blood for
the haemolytic serum is taken from the rabbit five days after the second
and last injection. He states that such a method not only gives a
high titer but avoids to a great extent agglutination difficulties. It
also is more stable as regards holding its original titer. We have had
better success with the following method than with Coca's. Inject in-
travenously Y± c.c. of 50% red-cell suspension and three days later
inject ^ c.c. of the same strength suspension. Several days later in-
ject i c.c. and withdraw seven days afterward.

THE WASSERMANN TEST

In the Wassermann reaction the rabbits are injected with sheep
red cells which have been washed twice with salt solution by aid of the
centrifuge. About five intraperitoneal injections with, on the average,
the quantity of red cells contained in 5 c.c. of sheep blood, given at in-
tervals of five days, gives a strong haemolytic serum if taken about
one week after the last injection.

12

178 PRACTICAL METHODS IN IMMUNITY

The standardization of the titer is similar to that for the human hsemolytic
amboceptor, except that 5% emulsion of sheep cells with 0.05 c.c. undiluted guinea-
pig serum is used instead of the %% emulsion of human cells and the o.i c.c. of 40%
guinea-pig serum.

The method is to take in a test-tube 0.2 c.c. inactivated human
serum (heated for twenty minutes at 56°C.), o.i c.c. fresh serum from
guinea-pig for complement, i unit antigen and 3 c.c. normal salt solu-
tion; then to incubate for one hour at 37°C. (An antigen unit is the
amount that will inhibit haemolysis of i c.c. of 5% emulsion of sheep
cells when mixed with 0.2 c.c. luetic serum and o.i c.c. guinea-pig
complement.)

There should likewise be absolutely no inhibition with 0.02 c.c. of serum from a
normal individual. The unit is made up to i c.c. with salt solution.

Then add 2 units of amboceptor and i c.c. 5% emulsion of sheep
red cells, shake and incubate for one hour. (The amount of haemolytic
serum that will haemolize i c.c. of a 5% emulsion of sheep red cells
to which 0.05 c.c. guinea-pig serum has been added, in one hour, is an
amboceptor unit.)

In our laboratory we use i c.c. of a i to 10 dilution of the clear stock acetone
insoluble antigen of Noguchi as the antigen unit.

In the preparation of the antigen originally recommended by Wasser-
mann, we finely divide the liver of a syphilitic foetus and extract it for
twenty-four hours with salt solution containing 0.5% carbolic acid.
Frequent shaking is required. The supernatant fluid is decanted and
centrifuged. This turbid fluid is pipetted off and kept in the ice-box in
a brown bottle for several days. This yellowish-brown opalescent fluid
is the antigen and is standardized so that i unit equals the amount
which will inhibit haemolysis of i c.c. of 5% emulsion of sheep red
cells when mixed with 0.2 c.c. luetic serum, o.i c.c. guinea-pig com-
plement and 2 units of amboceptor.

The same technic is employed with the control test-tube except that
the antigen unit is not put in.

The Noguchi method has been stated to give a positive reaction with nonsyphilitic
sera in about 7% of cases. The Wassermann a negative result in about 9% of syph-
ilitic sera. These figures show the advantage of checking one against the other.

One objection to the Wassermann test is that a majority of human
sera show native antisheep amboceptors and in some instances the
amount of this constituent may be so great, when added to the 2 units

EMERY'S TECHNIC 179

of amboceptor from the serum of the sheep cell immunized rabbit, as
to cause haemolysis with a syphilitic serum. Simon recommends
treating the sera to be tested with sheep cells in order to fix these
native amboceptors. Centrif ugalizing, the sheep cells with the attached
amboceptors are thrown down, leaving the clear supernatant serum
free of these amboceptors.

There seem to be certain sera when with a clinical history of syphilis we obtain a
positive Wassermann with unheated serum and a negative one with inactivated
serum. In order to obtain information with the same serum heated and unheated
I would recommend, when it is inconvenient to carry out the original Wassermann
technic, to employ the Noguchi technic with inactivated serum and the Emery
technic with fresh unheated serum. In any case when serum cannot be tested within
twenty-four hours it should be inactivated, as unheated serum tends to become
anticomplementary.

EMERY'S TECHNIC FOR THE WASSERMANN TEST

Owing to technical difficulties with the method of making and
employing the antigen and amboceptor features of the original Emery
test, I have retained the principle of the test but substituted the
reagents prepared in exact accordance with Noguchi's directions.

Briefly stated, the principle of Emery's test consists in the employment of fresh
human serum for supplying complement and the primary incubating of the haemolytic
system (human red cells and rabbit serum immune to human red cells) at the same
time as the incubation of the antigen and serum but in separate tubes. Then in the
second period of incubation to add these "sensitized" cells to the serum antigen
combination.

In Noguchi's method all reagents are incubated together. in the
first period with the exception of the amboceptor paper (dried serum
of rabbit immune to h iman red cells), which is not added until the
period of incubation for complement binding is completed and the
second incubation commenced. Time is saved in the Emery technic,
inasmuch as the red cells are already sensitized by the haemolytic
amboceptor when added to the tubes, and haemolysis shows itself
almost immediately in tubes where the complement has not been
absorbed by the antigen through syphilitic antibodies.

Noguchi has called attention to the fact that protein constituents of certain
aqueous or alcoholic extracts may have the power to fix complement through certain
intermediaries existing in fresh serum which, however, does not obtain for inacti-
vated sera (sera heated to s6°C. for fifteen minutes).

i8o

PRACTICAL METHODS IN IMMUNITY

Pure lipoidal substances as contained in Noguchi's acetone insoluble antigen,
however, do not act in this way.

Consequently by using such an antigen we eliminate the objection to employing
fresh human serum in the test for syphilitic antibodies.

As giving more uniform haemolytic results and as being more stable and easier
of employment, I have made use of Noguchi's directions for taking up the serum of
the rabbits, immunized to human red cells and his method of standardizing this
"amboceptor" paper. In practice, I measure off the length of paper correspond-

CONTROL

U. 40 I tobO
ANTIGEN ANTIGEN ANTIGEN ANTIGEN ANTI&EN ANTIGEN

5

FIG. 49. — i. Capillary pipette being graduated by drawing up i and 4 drops
from a watch-glass, (a) Blue pencil mark of i drop or i volume, (b) Mark of
volume of 4 drops. 2. Graduated centrifuge tube containing sodium citrate normal
salt solution. 3. Tube with 10 amboceptor units in i c.c. of salt solution. 4. Mix-
ture of i volume 20% emulsion red cells and 4 volumes inactivated amboceptor
solution. 5. Small glass tubes for Emery test. 6. Method of transferring from
tube to tube. 7. Making a Wright U-tube — the end "a" to be used as a capillary
pipette.

ing to 10 Noguchi units and dissolve the dried serum in such paper in i c.c. of salt
solution. This make a satisfactory and uniform substitute for the sterile immune
serum used by Emery.

Method: i. Take blood from the finger or ear in a large Wright U tube (Y± inch
in diameter). Place in 37°C. incubator for fifteen minutes (to increase yield of
serum) and then centrifuge.

Of course wheli the Noguchi test is being made at the same time we
use the serum of the blood taken from a vein in the arm.

EMERY'S TECHNIC FOR WASSERMANN

181

2. Graduate a capillary pipette for i volume and 4 volumes.

There is the greatest variation in the size of a drop delivered by a capillary pipette,
this difference in size not only occurring with varying diameters of the capillary
tube but with position of tube, thus a tube with i mm. diameter and held horizon-
tally will deliver about 16 drops of serum to the c.c. If held vertically about 32. A
fine capillary pipette, such as is used by Noguchi will deliver, when held vertically,
50 drops to the c.c., or 0.02 c.c. to the drop. It is therefore better to standardize
with known amounts delivered from a graduated pipette, using J-^Q c-c- as the un^
for i volume.

FIG 50— i. Copper water bath 12X12X8 inches, (a) Thermometer to show
38°C (b) Tubes containing antigen dilutions, (c) Tube containing hamolytic
system incubating along with the antigen tubes. 2. Ordinary rice cooker with copper
holder for test-tubes.

3. Into each of a series of small test-tubes put 4 volumes of normal salt solution.
(These tubes are most conveniently made by breaking off 2^- to 3-inch lengths of
24-inch soft glass tubing and then fusing one end in the flame to make a small test-
tube.)

Make a distinguishing mark, e.g., X, on end of tube with blue-wax pencil and i
this tube to hold control. Mark the other tubes I, II, III, and so on. When differ-
ent sera are to be tested they may be distinguished by lines either above or below,
or with circles; also marks with red-wax pencil may be used.

4. Make a i to 20 dilution of stock antigen solution in salt solution.

To Tube I add 4 volumes of i to 20 antigen, thus making 8 volumes of i to 40

1 82 PRACTICAL METHODS IN IMMUNITY

antigen in Tube I. Mix thoroughly by manipulating bulb of pipette. Then trans-
fer 4 volumes of the i to 40 from Tube I to Tube II, and so on through the series.
When the dilution in the last tube has been made throw 4 volumes away.

The 4 volumes of dilution of the antigen in the respective tubes will then be:
In Tube I, i to 40; in Tube II, i to 80; in III, i to 160; in IV, i to 320, and so on.

5. Add i volume of serum to be tested to control tube X, and to each of the tubes

I, II, III, etc., in succession. (If the serum be added to the antigen tubes before the
control tube, antigen might be carried over to the control.)

NOTE. — If the serum has been inactivated restore complement by adding i volume
of a 40% fresh guinea-pig serum. Also use 2 volumes of this inactivated human
serum instead of i.

6. Incubate at 38°C. for thirty minutes. This allows syphilitic antibody, if
present, to bind complement.

7. As soon as the above mixtures have been made and put in the incubator
prepare the "haemolytic system" by adding i volume of 20% emulsion of washed
human red cells to 4 volumes of solution of amboceptor paper (10 Noguchi units of
amboceptor paper dissolved in i c.c. of salt solution. Thus of a paper of which 4 mm.
was the unit we should cut off about 40 mm., place in test-tube and extract the dried
serum with i c.c. of salt solution), and place this haemolytic system in incubator
alongside the tubes already there. To obtain the washed red cells allow 4 to 10
drops of blood to drop into a graduated centrifuge tube containing salt solution to
which has been added i % of sodium citrate to prevent coagulation. After shaking,
centrifuge. Pour off supernatant fluid, replace with salt solution, again shake and
centrifuge — this sediment of red cells is to be diluted with 4 volumes of salt solution
(20% emulsion). (Incubation hastens sensitization of the red blood cells. Agglu-
tination of red cells may occur with certain haemolytic sera. The immunization of
the rabbits with small doses intravenously (Coca's method) tends to prevent this
interfering factor. However, frequent shaking is usually sufficient to break up the
agglutinating masses of red cells).

8. At the expiration of thirty minutes from the commencement of incubation
for complement binding, add i volume of haemolytic system to each of the tubes, I,

II, III, etc., in the order of antigen dilution.

9. Finally, after washing pipette in salt solution, add i volume of haemolytic
system to control in tube X. (If the haemolytic system should be added to the con-
trol tube before the antigen tubes, complement from the control tube might be
carried over to the antigen tubes.)

Shake each tube thoroughly. Allow them to incubate for a few minutes. Then
examine tubes I, II, III, etc., for haemolysis. The control should, of course, show
haemolysis. The antigen tubes should show a white, supernatant fluid over the in-
tact red-cell sediment in the tubes with the low dilutions and even in the highest
dilutions, where the serum is strongly positive. In a weakly positive serum, in-
hibition of haemolysis may only show in the first tube and haemolysis show in those
tubes having higher dilutions of antigen.

It will be noted that the reagents are made in accordance with
Noguchi's directions. Even in those cases where fresh guinea-pig
serum is employed to replace complement, absent from the inactivated

USE OF SENSITIZED CELLS FOR NOGTJCHI TECHNIC 183

human serum tested, we employ the 40% solution used in Noguchi's
technic.

Experiments have shown that one volume of serum contains almost invariably
sufficient complement to haemolyze the red cells present. As a matter of fact in 95%
of sera one-half this amount would suffice.

It will be noted that the amboceptor and antigen content of the
i to 80 tube is proportionate to that used in the Noguchi test.

NAVAL MEDICAL SCHOOL MODIFICATION OF NOGUCHI'S TECHNIC

There is evidence for believing that the heating of the serum to be
examined not only destroys about 75% of its total syphilitic antibody
content but may also destroy thermolabile substances which may be
of importance in bringing about a positive reaction. At any rate I
have had experience with sera where, with an absolutely clear diagnosis
of syphilis followed by response to therapeutic measures, there has
been a negative reaction with inactivated serum but a positive one with
unheated serum.

For this reason I would advise that the Noguchi method be carried
out with inactivated serum and an Emery test also made using un-
heated serum.

The Emery test is also of value as showing a quantitative relation. At the same
time I am convinced that it is more conservative to make a diagnosis of syphilis only
with inactivated serum, reserving the utilization of findings with unheated sera for
effect of treatment on syphilitics and for such cases as the symptomatology and
history of the case would indicate that a positive reaction should be obtained.

No injury is conferred on a patient by a negative Wassermann be-
cause in the presence of other data treatment will not be withheld.

A modification of the Noguchi technic, along the Emery lines, which saves time
and makes readings sharper is to employ sensitized red cells. To do this add to the
drop of patients serum and 40% guinea-pig complement and antigen in tube la, %
c.c. of salt solution. Also to tube ib, containing the serum and complement but
without antigen add 14 c.c. salt solution. Incubate for absorption of complement.
So soon as these tubes are placed in the incubator prepare a mixture of i% washed
red cells, according to the amount to be used in the number of tests to be carried
out, and add 2 units of amboceptor paper for each Y% c.c. of this red-cell emulsion.
Incubate along with the other tubes. When the period for the primary incubation is
completed add to each tube K c.c. of the emulsion of sensitized red cells. ^

Incubate again and readings can usually be made in ten to fifteen minutes and
give unusually clear-cut readings.

184 PRACTICAL METHODS IN IMMUNITY

General Considerations. — Cherry thinks anticomplementary bodies are found
during chloroform anaesthesia. If the antigen should also have anticomplementary
action the total might give a negative result.

By heating the serum for half an hour at s6°C. (inactivation) the positive results
reported to have been obtained in certain cases of cancer, nephritis, scarlet fever,
leprosy and tuberculosis may be avoided; the syphilitic antibody alone being more
thermostable. The thermostability of serum of inherited syphilis is the highest —
that of primary syphilis the least of luetic sera.

McDonagh states that in the primary stage the Wassermann is positive in 40% of
cases. In secondary cases 97% give positive results when treatment has not been
instituted. In tertiary syphilis about 70% are positive.

In 268 cases at the medical clinic of Johns Hopkins Hospital, Clough
failed to obtain a positive reaction in 99 cases which were negative
clinically.

In 45 cases of syphilis he obtained 73% of positive results. Ex-
cluding cases which had received thorough treatment 82% were
positive. Tabes gave 40% and general paresis 100%. In five cases
of primary syphilis four gave positive reactions. Kolmer gives 96%
positives for untreated active tertiary syphilis with 75 to 80% for
latent tertiary syphilis. In untreated congenital syphilis of children
over one year of age, 97 to 100%. Comparing the luetin reaction
with the Wassermann, Noguchi gives 80% for tertiary and 70%
positive for congenital syphilis.

Based upon the observation of Bauer, that human serum contains haemolytic
amboceptors for sheep corpuscles, and of Hecht, that the complement normally
present in human serum would suffice without the addition of guinea-pig serum
complement, the following method of Fleming is easy of application, but not
recommended.

For the test we use:

1. Alcoholic extract of rabbit's heart, made by washing the recently removed
heart with salt solution to remove all blood. Cut into small pieces and grind in a
mortar with sand and for every gram of heart add 5 c.c. of 95% alcohol. Keep the
mixture at a temperature of 6o°C. for two hours and filter. This is the stock solu-
tion. For use dilute it 10 times with normal salt solution.

2. A 5% emulsion of washed sheep red cells, prepared as for the Wassermann test.

3. Suspected and control sera.

With a capillary bulb pipette take up i part of serum and 4 parts of the heart
antigen, mix on a glass slide, again draw up into the capillary pipette and,
leaving a separating air space, next draw up i part of 5% emulsion of sheep red
cells. Then seal off tip of pipette and incubate at 37°C. for one hour. Now file
off tip and mix the red cells with the serum and antigen and again draw up into the
capillary pipette and incubate a second time for two hours. Haemolysis or the
reverse is shown in the fluid overlying the cell sediment. Various controls should
be made using normal and known syphilitic sera; also with normal salt instead of
serum.

BACTERIAL COMPLEMENT FIXATION TESTS 185

The objections to methods using human serum for complement are
i. the great variation in the complement content of different human
sera; 2. human complement requires about 10 times as much ambo-
ceptor as guinea-pig complement and is less sensitive to fixation, and
3. the statement is made by some workers that while homologous
complement and amboceptor may be efficient yet the complement of a
serum will not act upon its homologous antigen. This is not true
because the complement of human serum invariably haemolyzes the
homologous antigen (human red cells).

The various precipitate tests that have been proposed are unreliable. The
precipitate reactions with bile salts give better results than with lecithin, this latter
showing positive results in almost one-half of non-syphilitic cases.

BACTERIAL COMPLEMENT-FIXATION TESTS

The two bacterial complement-fixation tests which are used as
routine diagnostic methods are those for gonorrhoeal and glanders
infections.

Of course similar tests may be made for typhoid, cholera, etc., but we have more
simple and practical methods in the use of agglutination or, in the case of typhoid,
blood culture procedures.

The two best known methods for preparing bacterial antigens are
the following:

1. Emulsify the growth on agar or starch agar (for gonococcus) in salt solution, as
described under preparation of vaccines. Heat the emulsion at 6o°C. for one or two
hours and then count the organisms as for vaccines.

For gonococcus tests we use an antigen with 4,000,000,000 organisms in i (?.c.
This may be used directly as antigen or it may be shaken up with glass beads for
several hours to complete disintegration. The antigen can be preserved by the
addition of y±% trikresol or Y2% phenol. For glanders one may use a seventy-
two-hour culture in glycerine bouillon, sterilized at 6o°C. for two hours and preserved
with 0.5% phenol.

2. Besredka and Gay prepare their antigen by precipitating the saline bacterial
emulsion, washed-off agar, with an equal amount of absolute alcohol. Then centrifu-
galize, pipette off supernatant fluid and dry the sediment in vacua over sulphuric
acid. The dried sediment is made into a 2% suspension with isotonic salt solution.
For use this stock solution is diluted. There are also methods in which the bacterial
sediment is frozen with carbon dioxide snow and then triturated with crystals of
sodium chloride so as to make an isotonic saline emulsion.

Bacterial sediments can also be dried in calcium chloride desiccators.

In carrying out bacterial complement-fixation tests we use an amount

1 86 PRACTICAL METHODS IN IMMUNITY

of antigen which will by its antigenic power alone fix complement or,
as is often stated, be anticomplementary.

Then use one-half this amount as the antigen content for the test.

The method of Noguchi, as previously described, but using one-half the anticom-
plementary dose of antigen, is satisfactory after experimenting with the proper
amount of inactivated serum of the patient to be examined.

For the Gonococcus fixation test it is most important to have antigen
prepared from a mixture of several strains of gonococci, preferably
10 or 12.

In our laboratory we have had sharper and more satisfactory readings by employ-
ing the Emery technic, placing in the first antigen tube an emulsion containing
4,000,000,000 organisms to the c.c.

It is also satisfactory to have the antigen in tube i so concentrated that it will
prove anticomplementary. Tube 2 would contain one-half this amount of antigen;
tube 3, one-fourth; tube 4, one-eighth; tube 5, one-sixteenth; tube 6, one-thirty-second;
tube 7, one-sixty-fourth. Some sera are strong enough in specific gonococcus anti-
body to bring about complement fixation in tube 8. Along with the test of the
patient's serum we should carry out tests with known negative and positive sera.

DETERMINATION OF OPSONIC POWER AND THE PREPARATION
OF VACCINES

In that which has been considered in the previous pages only the
theories of Ehrlich have been brought out. In order to understand the
problems involved in the study of opsonins the phagocytic theory of
immunity brought forward by Metchnikoff must be studied. Ehrlich's
views would seem to hold with diseases where there is an increase in
bacteriolytic or antitoxic power of the serum while in such diseases, as
are caused by pathogenic cocci, the pjiagocvtic element js operative as
there is an absence of bacteriolvtic power in the serum of the person
with the infection.

There are two kinds of phagocytes, the microphages (represented by the poly-
morphonuclears) which on phagolysis or disintegration give off microcytase, a
bactericidal substance. Cytase is the same as complement or alexine.

The microphages are chiefly bactericidal while the macrophages, represented by
the large mononuclears of the blood and fixed connective-tissue cells^exert their
action on protozoa or animal cells. .

(t)

Phagocytes may either act by m^estin^ j^cteria and destroying

them intracellulary or they may as a result of phagolysis bring about
bacteriolysis exr.ra.r.p.11n1a.rlv. According to Metchnikoff the intra-

OPSONIC INDEX 187

cellular bacteriolysis explains why an individual may possess immunity
and yet his serum fail to show any bacteriolytic power.

The following modification of Irishman's method takes very little time and skill
and is applicable in the determination of the organism concerned in an infection, as
in Wright's method. The control of vaccine treatment by taking opsonic indices
from time to time does not seem to have met with much favor in this country — the
sources of error being as great if not greater than ordinary variations in the opsonic
index during the negative and positive phases.

Method. — We start with a i % solution of sodium citrate in salt solution. With
this emulsify a twelve- to twenty-four-hour agar slant growth of the organism to be
tested using 6 to 8 c.c. of the citrated salt solution. The bacterial emulsion is now
poured into a bottle or sealed off in a test-tube and shaken thoroughly in a shaker or
by hand. The emulsion is then centrifuged to throw down the bacterial clumps and
the supernatant slightly turbid bacterial suspension pipetted off. If working with a
dangerous pathogen it is advisable to kill the organisms as in making vaccines.

Now with a capillary bulb pipette so graduated that the one volume mark con-
tains about o.i c.c. we draw up one volume of citrated salt solution. Then having
made a break with an air column, we take up one volume of the patient's blood.
Again make an air break and draw up one volume of the citrated salt solution
bacterial emulsion. The 3 volumes are then immediately forced out into a
small test-tube, made from 3 inches of ^6-inch glass tubing, as shown in the
Emery technic for the Wassermann. The citrate prevents coagulation of the blood
and the contents of the tube are well mixed by drawing up and ejecting with the
capillary bulb pipette. Incubate this small test-tube at body temperature for fifteen
minutes, shaking the contents once or twice during the incubation period. Exactly
at the expiration of the period of incubation (usually fifteen minutes although at
times ten minutes or thirty minutes may be desirable) place the tube in a centrifuge
and throw down the cell sediment. Next pipette off the supernatant fluid and then
plunge a finely drawn out capillary pipette to the bottom of the tube and draw off
the greater part of the sediment at the bottom. This consists largely of the red
cells the leukocyte layer on the surface being undisturbed.

Now mix the remaining cell sediment and smear out on a slide or preferably
between two cover-glasses as in Ehrlich's method. The smear is fixed by burn-
ing off a film of alcohol or with methyl alcohol and stained with dilute carbol
fuchsin or methylene blue. The granule staining with Wright's stain makes it
slightly confusing.

A second similar preparation but using blood from a normal person as a control
is then made. Counting the phagocytized bacteria in a given number of poly-
morphonuclears, we obtain an average number of bacteria phagocytized per cell.
Repeating the count with the control or normal blood, we likewise have the average
number of bacteria taken up per cell. Dividing the patient's average by the
normal average, we have the opsonic index. If the average for 50 of the patient's
cells was 8 and that of the control only '4, the patient's index would be 2, or twice
the normal. The practical value of this test is that where two or more organisms
are on a plate from a body fluid we may ascertain the causative organism by
noting marked variation from the normal in the patient's opsonic index for that

1 88 PRACTICAL METHODS IN IMMUNITY

particular organism and not for the other organism. This variation may be of
the nature of a high or low opsonic index.

METHOD OF WRIGHT FOR OBTAINING OPSONIC INDEX

While other observers had previously noted the presence of sub-
stances in immune sera which so acted on the bacteria that phagocytosis
was made possible, yet it was to Wright and Douglas, in 1903, that the
existence of this factor in phagocytosis was brought forward and the
estimation of such substances made practicable.

To this substance the name opsonin was given — the Greek word from which it is
derived indicating preparation of the food — that is, the opsonin so alters or sensitizes
the bacteria that they can be engulfed or phagocytized by the polymorphonuclear
leukocytes (the microphages of Metchnikoff) . About the same time Neufeld and
Rimpau noted the presence of a substance in immune sera which so acted on bacteria
as to prepare them for phagocytosis. Their designation "bacteriotropic substance"
is practically synonymous with opsonin.

In 1902 Leishman introduced the method of determining the "phagocytic index."
By taking i part of blood and impart of an emulsion of the bacteria in question and
keeping the mixture in a moist chamber at body temperature for a standard time,
as fifteen to thirty minutes, and then spreading the blood-bacteria mixture and
staining the film with Leishman or Wright's stain he counted the number of bacteria
in a certain number of polymorphonuclears, and by dividing obtained the average
number per leukocyte of bacteria phagocytized.

The Wright technic for determining the phagocytic average, and
from this the opsonic index, is as follows:

Blood is taken from the patient and at the same time from a normal individual,
or preferably the blood of several normal individuals is pooled. This blood is best
collected in a Wright's tube, although it may be received in a small test-tube. After
coagulation and separation of the serum, the serum is ready for use.

The next step is to prepare the leukocyte gmulsion. For this we fiU a centri-
fuge tube with normal saltsolution, to which has been added i% 'sodium, citrate
— the latter to prevenTcoagulation. Then having pricked a finger congested by a
constricting rubber band, from 15 to 20 drops of blood are added to the citrated
salt solution, and the mixture thoroughly shaken. After centrifugalization for about
five minutes the red corpuscles will be thrown to the bottom of the tube with the
leukocytes forming a superimposed layer. In order to free the leukocytes entirely
from serum admixture, the supernatant citrated salt solution is pipetted off, and a
fresh tubeful of salt solution is added to the blood-cell sediment. Again shak-
ing, we centrifuge, obtaining for a second time a sediment of blood cells with the
leukocytes in the superimposed layer. In some laboratories the washing in salt
solution is again repeated, but for all practical purposes two washings as described
above suffice.

The superimposed layer of vyhite cells may now be pipetted off from the heavier

VACCINES 189

red cells (of course, containing a large admixture of red cells) to be used as a leuko-
cyte cream — or by slanting the centrifuge tube we can pipette off the proportion
of the leukocyte mixture needed from the bottom, sides or top of the slanted layer
of blood cells.

Having prepared our leukocyte j;mulsion. and the serum from the normal indi-
vidual as well as that from the patient, it only remains to prepare our bacterial
emulsion. For bacteria in general, with the exception of tubercle bacilli, we simply
take up a small loopful of a young agar culture (eighteen hours or less), and emulsify
it uniformly with salt solution, added by degrees until the suspension amounts to
Yz to i c.c., and giving a faint turbidity. To thoroughly distribute and especially
to break up clumps repeated suction and ejection with a capillary pipette provided
with a rubber nipple is satisfactory.

The presence of clumps in a bacterial emulsion invalidates the estimation of
phagocytosis, for the reason that a leukocyte will take up a clump of twenty or more
bacilli as readily as one separate organism.

Having at hand (i) the suspension of leukocytes, (2) the bacterial emulsion,
and (3) the sera of the patient and the normal individual, we are ready to proceed
with the test.

Using a capillary bulb pipette with a pencil mark to indicate i volume we draw
up to the mark (i) the leukocyte cream. Then wiping off the tip of the pipette we
draw up this volume of leukocyte emulsion about % inch to make an air break
between this and (2) i volume of the bacillary emulsion. Again making an air
space we draw up (3) the serum of the normal individual. This gives three columns
in the capillary tube with intervening breaks of air. We next eject the three con-
stituents into a watch-glass and thoroughly mix them by alternate suction and ejec-
tion with the tube and nipple. When mixed we draw the mixture up into the same
capillary tube, seal off the capillary end in the flame and put in an incubator for
exactly fifteen minutes.

We next repeat the process identically except that the patient's serum is used in-
stead of that of the normal individual.

These tubes having been kept at the same temperature for the same length of
time are then taken out, the contents blown into a watch-glass, mixed thoroughly
a second time, and then a smear is made — a drop of the mixture being deposited
on a very clean slide and the smear made by a second narrower slide (by cutting off
the corner of the slide) which is drawn along in a zigzag way. The smears are then
stained (Leishman's or Wright's blood stain or Ziehl-Neelson's for tubercle bacilli)
and the number of the bacteria in from 50 to 100 leukocytes counted. This
number divided by the number of cells gives the phagocytic average.

The phagocytic average of the patient's tube divided by that of the normal in-
dividual's tube gives the opsonic index. Thus, in counting 100 cells we find 500
phagocytized cocci in the patient's tube, giving an average of 5, and in the normal
individual's blood we get 1000, an average of 10. Then the opsonic index would be
5 -f- 10, or 0.5.

PREPARATION or VACCINES

It has been found satisfactory to make use of stock vaccines in-
gonorrhceal and tuberculous affections. In treatment of tuberculosis

1 go ' PRACTICAL METHODS IN IMMUNITY

Wright prefers Koch's T. R. or Neu Tuberculin in doses of from J
to J^oo rng. Some prefer Koch's more recent bazillen emulsion. In
case of other infections, however, and preferably with gonorrhceal in-
fections, the causative organism should be isolated from pus, sputum,
urine, blood, or other material from patient (autogenous vaccine).

In the making of vaccines all media and apparatus should be sterilized with
scrupulous care to avoid the danger of tetanus infection. Having isolated the organ-
ism, it is inoculated upon one or more agar slants, and after a growth of from five
to seven hours with streptococci and pneumococci, or with eighteen hours for staphy-
lococci and colon, the growth on these inoculated slants is taken up with salt solu-
tion, thoroughly shaken up in the diluting solution and standardized. (Esmarch
roll of nutrient agar may be inoculated for growing cultures for vaccines.)

The most practical way is to gently rub off the growth on the agar in about i or

2 c.c. of salt solution with a platinum loop or sterile cotton swab. Then pour the
bacterial emulsion into a sterile test-tube and repeat the process with three to five
agar slants, until we have from 6 to 10 c.c. of the emulsion in the sterile test-tube.
By heating to melting-point in the flame a piece of glass tubing and attaching it to
the rim of the test-tube (also melted), we have a handle with which to draw out the
test-tube when heated about i inch from the mouth in a blowpipe flame. Drawing
this out, we let it cool, and then filing the constricted portion we break it off and seal
it in the flame. By shaking up and down vigorously for five to fifteen minutes, or
preferably in a mechanical shaker the bacteria are distributed evenly in the salt
solution. A piece of platinum wire, twisted into corkscrew shape, and fused in the
drawn-out end of the containing test-tube helps in breaking up the bacterial emulsion
and is a great aid in the preparation of streptococci or diphtheroid vaccines.

The sealed test-tube is then placed in a water-bath at 6o°C. and heated at this
temperature for one hour. Again shake. The constricted sealed end is again filed
off and a few drops shaken out in a watch-glass for standardization, and at the same
time a few drops are deposited on an agar slant as a test for sterility. (Incubation
for twenty-four to forty-eight hours should not show growth.)

Wright found that by taking a definite quantity of blood and a
similar quantity of bacterial emulsion, mixing the blood and bacterial
emulsion, then making a smear and staining, it was possible to de-
termine the ratio of bacteria to red cells, and from this the number of
bacteria per cubic centimeter could be determined. For example, if
we find three bacteria to each red cell we should have 15,000,000
bacteria to i c.mm. (There being 5,000,000 red cells to the cubic milli-
meter.) As i c.c. is 1000 times greater than i c. mm., there would
be 15,000,000,000 bacteria in each c.c. of such an emulsion, or vaccine,
as it is termed.

The standardization made with a haemacytometer is best done by drawing up
the vaccine to 0.5 with either the red or white pipette, according to concentration,

DOSAGE OF VACCINES igi

and then sucking up one- twentieth of i% dahlia in i% formalin to n or 101.
Allow the bacteria to settle on the shelf for ten minutes before counting. Count as
in making a red count.

The use of a piece of amber glass in front of an incandescent light
enables one to pick up the bacteria more satisfactorily as well as to
differentiate bacteria from debris. A counting chamber with a depth
of J^o mm. is to be preferred to the ordinary % o mm. chamber as one
can begin the count after about five minutes time for settling.

A satisfactory diluting fluid is that recommended by Callison. It is : Hydrochloric
acid 2 c.c., Bichloride of mercury (i to 500 aq. sol.) 100 c.c., and sufficient i%
aqueous solution of acid fuchsin to color the diluting mixture a deep cherry red.
The diluting fluid should then be filtered. The bichloride forms an albuminate
on the surface of the bacteria which promotes rapid sedimentation and the fuchsin
stains the bacteria.

Having determined the strength of the stock vaccine, we should
prepare a dilute vaccine for injection.

This is most conveniently carried out by filling vials with 50 c.c. of salt solution,
plugging with cotton, then sterilizing in the autoclave. A sterile rubber cap is now
drawn over the mouth of the vial. Sterility is insured by plunging the rubber cap
and neck in boiling water. If the stock vaccine showed 5,000,000,000 bacteria per
c.c. and we desired to have a vaccine containing 200,000,000 bacteria per c.c., it
would be necessary to draw out 2 c.c. of the salt solution by means of a sterile syringe
needle inserted through the rubber cap and replace it with 2 c.c. of the bacterial
emulsion. Example: In introducing 2 c.c. of a vaccine containing 5,000,000,000
bacteria per c.c., we throw in 10,000,000,000 bacteria in a volume equal to 50 c.c.
Then each c.c. of the 50 c.c. in the bottle would contain 10,000,000,000 divided by
50 or 200,000,000 in each c.c. If we only want a vaccine containing 100,000,000 per
c.c. we should only add i c.c. We now add Y±% of trikresol to the vaccine in order
to insure sterility. (Introduced with syringe, inserting needle through rubber cap.)
The syringe is best sterilized by drawing up vaseline or olive oil heated to i5o°C.,
and the neck and rubber cap of the bottle in boiling water. We now draw up the
desired dose of bacteria. If glass syringes are used, simply boiling in water suffices.
One can purchase ampoules which are sterilized and filled with a standardized
emulsion. They are sealed in the flame and labelled with the bacterial content
per cc.

The ordinary doses are: For gonococci, streptococci, pneumococci,
and colon vaccines, 5,000,000 to 50,000,000. For staphylococci

200,000,000 tO 1,000,000,000.

Wilson gives the following minimum and maximum doses expressed in millions:
Streptococcus, 6 and 68.
Gonococcus, 45 and 900.
Meningococcus, 300 and 900.

I Q2 PRACTICAL METHODS IN IMMUNITY

M. mclilensis, 700 and 1400.

B. coli, 1 6 and 240.

B. typhosus (treatment) 100 and 250.

B. typhosus (prophylaxis) 500 and 1000.

B. pyocyanens, 34 and 1000.

B. pneumonia, 44.

Staphylococci, 150 and 900.

B. tuberculosis, Hoooo to /^oo rag.

Sensitized Vaccines. — These are prepared by treating the bacteria
with the specific serum and cause less reaction than ordinary vaccines.
To prepare them the bacterial growth is treated with its antiserum
for three hours, thrown down in the centrifuge and the supernatant
serum removed. After washing in salt solution they are emulsified in
salt solution and killed at a temperature of 56°C. for one hour. Besredka
has used living sensitized bacteria in typhoid.

The question of the best method of preparing vaccines for prophylactic use is still
unsettled. The greatest difficulty has been experienced in making vaccines of the
Shiga bacillus on account of the great toxicity of such preparations.

Thompson has recently carried out some very important experiments at the
Lister Institute.

He worked with vaccines heated to s6°C. for one hour using ordinary methods as
well as organisms sensitized by treatment with specific serum. In another series
of vaccines he sterilized ordinary cultures as well as sensitized ones with 0.5%
carbolic acid in normal saline. He found that sterilfzation by heat not only destroyed
much of the immunizing power of the vaccines, but that such vaccines, whether of
ordinary bacterial emulsions or of sensitized organisms, showed great toxicity upon
their being injected and the heated sensitized ones were somewhat more toxic than
the nonsensitized organisms. Dean has used "eusol" for Shiga vaccines.

On the whole it would seem that sterilization with %% carbolic or Y±% trikresol,
using ordinary bacterial emulsions, is better than other methods.

Of course living organisms subjected to their specific serum have been recom-
mended in the case of typhoid but such methods are certainly not devoid of danger
and are not to be recommended for the present.

ANAPHYLAXIS

This is a term which indicates the opposite of prophylaxis. It was
noted that after a period of incubation of at least ten days a second
injection of horse serum produced symptoms of respiratory embarrass-
ment, convulsions and, at times, death. The primary injection had
during the period of incubation sensitized the cells to this particular
proteid. Extremely small amounts of serum will sensitize (o.oooi c.c.).
For anaphylaxis production much larger amounts are required; from

ANAPHYLAXIS

193

o.oi to 0.3 c.c. when given intravenously or intracardially and i or 2 c.c.
when given subcutaneously.

This phenomenon of sensitization in the case of rabbits bears the
name of Arthus, and as applied to guinea-pigs sensitized with diphtheria
antitoxin sera the name Theobald Smith, and it is stated by Muir
and Ritchie that active research as to anaphylaxis may be said to date
from the discovery of the phenomenon of Theobald Smith.

Rosenau and Anderson working with guinea-pigs showed that small
doses were efficient for sensitization, that the condition was trans-
missible from mother to offspring and that a second animal could be
sensitized by being injected with the serum of a sensitized animal.

This group of symptoms, the so-called anaphylactic shock, which is apt to set in
within a few minutes after the second injection, is often preceded by restlessness
and great excitement and together with the dyspnceic manifestations such as cough-
ing and rapid breathing, there is cardiac weakness and great fall of blood pressure.
The more serious symptoms as convulsions and at times death in from a few minutes
to an hour are more apt to appear after intracerebral injections than after intra-
peritoneal. Subcutaneous injections are least apt to produce anaphylactic symp-
toms. Our attention to this phenomenon commenced with the study of "serum
sickness" or "serum disease." In this an erythematous rash or urticaria associated
with more or less oedema comes on after eight to twelve days from the time of the
first and only injection of horse serum. It is supposed to be due to the fact that
some of the serum originally injected remains unchanged in the tissues so that
when the sensitization takes place there is present and at hand the same foreign
proteid to bring about anaphylactic symptoms.

Immunization against anaphylaxis is possible by repeating injection of the
sensitizing serum or proteid during the period of incubation. When a sensitized
animal recovers from the second injection it is afterward immune to anaphylaxis.
This is termed antianaphylaxis.

It is important to note that this hypersusceptibility appears to be
very rarely of importance in the matter of the administration of a
second injection of diphtheria antitoxin after the period of anaphylactic
incubation.

As a rule the death or untoward effects of the injection of serum are
in cases of status lymphaticus. Cases in man do occur, however, but
with extreme infrequency, in which within a few minutes after the
only injection of serum the patient becomes restless, shows symptoms
of cardiac and respiratory embarrassment and may be dead in a very
short time.

According to Rosenau and Anderson individuals who have asthmatic tendencies
as well as those who have had serum injections ten to twelve days or longer prior
13

I94 PRACTICAL METHODS IN IMMUNITY

to the second injection should be considered as possible subjects for anaphylactic
shock.

Vaughan recommends that when this is to be feared one should only give about
o.i c.c. of the serum and after an interval of two hours, provided no untoward
symptoms set in, to give the full amount of the injection. Besredka advises heating
the serum to 56°C. as a guard against anaphylactic shock.

Allergy. — The condition of hypersusceptibility or anaphylaxis is at
times termed allergy. Thus in a person who has been successfully
vaccinated a reaction shows at the site of inoculation within twenty-
four hours which does not appear in the nonimmune person for a period
two or three times as long. The diagnostic tests with tuberculin and
luetin are hence often referred to as allergic reactions.

Toxic Protein Split Products. — It may here be stated that some in-
vestigators are of the opinion that our views not only as to immunity
but as to the essential nature of infectious diseases may be later on
found to rest in production of anaphylaxis. According to Vaughan
and others the parenteral introduction (hypodermic as opposed to
alimentary tract or enteral introduction) of foreign proteids excites the
formation of specific ferments in the cells and fluids of the animal
injected. After the ferment is formed a second injection of the same
proteid activates the ferment which splits up the proteid into a poison-
ous and nonpoisonous portion, the former causing the symptoms of
anaphylaxis or disease in the case of the poisonous split proteid of
bacterial pathogens.

The name anaphylactine has been applied to the sensitizing substance
produced during the period of incubation, and anaphylatoxin to the
poisonous part of the split proteid.

• It has been proposed to employ this phenomenon as a diagnostic measure. By
taking the serum of a tuberculous patient, which would contain the sensitizing sub-
stance, and injecting it into the peritoneal cavity of a rabbit, the animal would be
sensitized and an injection of tuberculin a few hours later would bring about the
phenomena of anaphylaxis in the rabbit.

This passive anaphylaxis, as it is termed, usually requires approximately twenty-
four hours for sensitization. This passive anaphylactic sensitization seems to dis-
appear in two weeks. It has been advised to passively sensitize guinea-pigs with
the serum of the person about to be injected and then twenty-four hours after inject
the guinea-pigs with the curative serum. If untoward results occur in the guinea-
pigs the patient should not receive the injection.

Recently Hagemann has found the following technic valuable in the diagnosis
of surgical tuberculosis. Guinea-pigs are inoculated intraperitoneally with tuber-
culosis cultures and by the end of the second week such pigs are sensitized. The
suspected material, as serous effusion, is injected intracutaneously and within twenty-
four to forty-eight hours a distinct swelling of the skin with a bluish-red center,

ABDERHALDEN TEST 195

which is surrounded by a porcelain white ring and outside of this a zone of inflam-
mation, shows a positive test.

THE ABDERHALDEN REACTION

According to Abderhalden, specific ferments of a protective nature
(abwehrfermente) appear in the circulation following parenteral injec-
tion of various materials and are different from ordinary antibodies.

The substance which causes the production of these specific ferments
is called the substratum instead of antigen, the usual designation of
antibody stimulating substances. The principle of the test is that
when serum containing such specific ferments is placed in contact, in
•vitro, with the substratum the latter is digested with the production of
soluble products, which can pass through a dialyzer and be recognized
in the dialysate. The special dialyzers are called thimbles and those
prepared by Schleicher and Schull are recommended.

About 0.5 gram of the substratum is placed in a thimble, which has been intro-
duced into a clean Erlenmeyer flask and covered with 1.5 c.c. of the patient's serum.

The thimble is then withdrawn, its open end closed by forceps, then thoroughly
washed with distilled water and again introduced into a clean Erlenmeyer flask
containing 20 c.c. of sterile distilled water. Toluol is then introduced into the
flask so as to cover the water around the thimble. These flasks are then put in
the incubator for eighteen hours and the dialysate tested for protein by the ninhydrin
test. For this one uses 0.2 c.c. of a i% solution of ninhydrin in water and adds
10 c.c. of dialysate and the mixture brought to a boil in a test-tube. A positive
reaction is a violet blue.

The biuret reaction may also be used.

The difficulties in the way of handling the thimbles and preparing the substratum
are so great that Abderhalden questions the competency of even experienced
serologists for the carrying out of the test.

The main objection to the test, however, is that it is affected quanti-
tatively so that using varying amounts of reagents one may obtain
contradictory results; thus it is possible to obtain ninhydrin reactions
with the serum of a male animal acting upon a placental substratum.

While the test is best known in connection with the recognition of
pregnancy it has also been employed for the diagnosis of malignant
tumors, etc.

In taking serum for the test it is necessary to withdraw the blood as
long after a meal as possible, preferably in the morning before breakfast.
The blood is taken into paraffin-coated centrifuge tubes and most
thoroughly centrifuged, so that it is absolutely free from red cells.
It is pipetted off and kept on ice.

196 PRACTICAL METHODS IN IMMUNITY

For the substratum the placenta should be obtained as soon after
delivery as possible or the malignant tumors as soon after operation
as is feasible. All connective tissue, blood, etc., should be removed
from the tissue from which the substratum is to be prepared.

The tissue is then cut up finely, put in a deep jar and washed several times with
distilled water. It is then boiled in a great excess of distilled water — 100 times as
much — and boiling continued about thirty minutes. This water is decanted and
the tissue added to fresh boiling water, repeating the process five or six times until
when placed in a dialyzing thimble no ninhydrin reacting soluble proteins can be
recognized in the dialysate by the blue color.

The sterile tissue is now placed in a jar, covered with distilled water and on top
of this toluol is deposited to maintain the sterility. To estimate the proper amount
of substratum to use in a test one must determine this using normal as well as
specific sera, recognizing the quantitative error of the test. The amount usually
called for is 0.5 gram.

Bronfenbrenner is of the opinion and has supported it by much
experimental work that it is not digestion of the substratum which
takes place but autodigestion of the specific serum when placed in
contact with its substratum, the products of such autodigestion being
of the nature of anaphylatoxins.

Anaphylatoxin Production Test. Bronf enbrenner's Modification of
Abderhalden's Test. — Bronfenbrenner found that if he treated the
serum of an •animal with a particular substratum and then injected
the autodigested serum intradermically he obtained a skin reaction.
This reaction, however, only obtained for homologous animals, those
of a different species not reacting satisfactorily.

He now reports satisfactory results with the following technic: About 2 c.c. of
the patient's serum is injected intraperitoneally into a guinea-pig, thus passively
transferring to the guinea-pig the specific substances of the human patient's serum.
The next day the guinea-pig is bled, its serum collected and placed on ice with a
suitable amount of substratum (placenta, bazillen emulsion, tumor tissue, etc.).

Eighteen hours later the serum is separated from the substratum by centrifugaliza-
tion, pipetted off and placed in the incubator for fifteen hours. At this time inject
0.05 c.c. of the autodigested serum, possibly containing anaphylatoxin, into the
shaven skin of a normal guinea-pig.

From twelve to twenty-four hours later a distinct skin reaction occurs at the site
of injection, provided the serum of the patient contained antibodies.

Instead of the skin reaction one may inject 0.5 c.c. of such autodigested serum
into the heart or vein of a normal guinea-pig and death will result of anaphylactic
shock.

If the patient's serum were negative the injection of as much as 5 c.c. would not
cause symptoms.

PART II
STUDY OF THE BLOOD

CHAPTER XIII
MICROMETRY AND BLOOD PREPARATIONS

MlCROMETRY

IN the examination of blood and faeces preparations, especially
when the identification of animal parasites is in question, there is noth-
ing that assists more than a knowledge of the measurements of the
object studied. The making of such measurements microscopically is
termed micrometry.

Micrometry is also indispensable in bacteriology and cytodiagnosis
as well as in animal parasitology.

The most practical way of making these measurements is with an ocular microme-
ter. These can be bought separately, or a glass disc (disc micrometer) with lines
ruled on it can be dropped into the ocular to rest on the diaphragm inside the ocular.
The ruled surface of this glass diaphragm should be placed downward. As was
stated in connection with the microscope, the image of the object is formed at the
level of the diaphragm rim inside the ocular, consequently the lines of the image cut
those of the lines ruled on the glass in the ocular. Once having standardized the
value of the spaces of the ocular micrometer for each different objective, all that is
necessary subsequently in measuring is to count the number of lines or spaces which
the image of the object fills and then, knowing the value of each space for that ob-
jective, to multiply the number of spaces by the value of a single space.

The Micron. — The unit in micrometry is the micron. This is usually
written ju and is the Ho 00 Part of a millimeter. There are 1000 microns
in a millimeter.

To standardize: For this purpose it is necessary to have a scale of known measure-
ments. The stage micrometers are usually ruled in spaces of o.i and o.oi mm. The
lines which are Ho mm. apart are consequently separated by a distance of 100
microns; those Koo mm. apart are separated by a distance of 10 microns.

Ocular Micrometer. — The ocular micrometer is usually ruled with
50 or 100 lines or spaces, separated by longer lines into groups of 5
and 10.

197

MICROMETRY AND BLOOD PREPARATIONS

Having brought the lines on the stage micrometer to a focus, we determine the
number of spaces on the stage micrometer which the 50 divisions of the ocular
micrometer cover. To distinguish the ruling of the ocular from that of the stage
micrometer, revolve the ocular with the fingers.

The tube length which is used at the time of standardizing must
always be adhered to in subsequent measurements.

FIG. 51. — Micrometry diagrams, i. Ocular micrometer with stage micrometer.
50 spaces of ocular micrometer cover two zoo-micron spaces and ten lo-micron spaces;
equal 300 microns. Each division on ocular micrometer equals 6-microns. 2.
Ocular micrometer subtending image of whip-worm egg. 9 spaces of ocular mi-
crometer cover whip-worm egg. Each space equals 6 microns. Whip-worm egg
equals 54 microns. 3. Ocular micrometer with ruling of hsemacytometer. 50
spaces of ocular micrometer cover space equal to width of 6 small squares 50X6 = 300
microns. Each division of ocular micrometer equals 6 microns.

Example: With a %-inch objective, the 50 rulings of the ocular micrometer
fill in fifteen of the ^0-mm. rulings (loojx) and three of the Hoo-mm. spaces (io/z).
Consequently the 50 spaces of the ocular cover 1530 microns (15 X 100 = 1500;
3 X 10 = 30). Then if 50 spaces equal 1530 microns, one space would equal
30.6 microns. With the J^-inch objective the 50 ocular spaces would cover about
three of the J^f o mm. (ioo/u) spaces of the stage micrometer. Then the 50 spaces
would equal 300 microns and one space would equal 6 microns.

The ruling of the slide of a Thomas-Zeiss haemocytometer will answer as well
as a stage micrometer. The small squares are %Q mm. square, consequently

BLOOD PREPARATIONS I 99

the distance between the lines bordering the small square is J^o mm. or 5°
microns.

Now, if with the ^-inch objective, the 50 lines on the ocular fill in the spaces of
six small squares, the length of such a space would be 50 X 6 = 300 microns. This
divided by 50 spaces would equal 6/*.

Should there be 100 spaces on the ocular micrometer instead of 50, it would
only be necessary to divide the length in microns of the ruled surface of the stage
micrometer covered by the 100 lines of the ocular micrometer by 100. The quotient
would give the value in microns of each space of such an ocular micrometer.

Filar Micrometer. — The most accurate instrument for measuring is the filar mi-
crometer. These are expensive. Measurements can also be made with the camera
lucida, but it takes considerable time to make the adjustments necessary, so that it
is not convenient. With an ocular micrometer one can make measurements of
blood-cells, amoebae, etc., in a few seconds — it only being necessary to slip in the
ocular micrometer.

Rule for determining the magnifying power of microscopic lenses: Measure the
diameter of the lens of the objective in inches — the approximate equivalent focal
distance is about twice the diameter. Dividing 10 by the equivalent focal distance
gives the magnifying power of the lens. This should be multiplied by the number
of times the ocular magnifies. Example: The diameter of the lens of the objective
was found to measure Y^ inch, the focal distance would then be about i inch. Divid-
ing 10 by i we have 10 as the magnifying power of the lens of the objective. If we
were using a No. 4 ocular, the magnifying power would be approximately forty.

BLOOD PREPARATIONS

To obtain blood, except for blood cultures, use either a platino-
iridium hypodermic needle which can be sterilized in the flame, a small
lancet, or a surgical needle with cutting edge.

When using such surgical needles it is a good plan to sharpen the cutting edge on
a fine-grained whetstone. Afterward the needle should be sterilized by boiling.
Sterilization of a needle in the flame blunts the cutting edge. A steel pen with one
nib broken off or the glass needle of Wright may also be used. To make a glass
needle, pull straight apart a piece of capillary tubing in a very small flame. Tap
the fine point to break off the very delicate extremity. Scarcely any pain attends
the use of such a needle. In puncturing either the tip of the finger or lobe of the
ear a quick piano-touch-like stroke should be used. The ear is preferable, as it is
less sensitive and there is less danger of infection. Before puncturing, the skin
should be cleaned with 70% alcohol and allowed to dry. It is advisable to sterilize
the needle before using it.

The first drop of blood which exudes should be taken up on the paper
of the Tallquist hsemoglobinometer, using subsequent ones for the blood
pipettes and smears. If it is necessary to make a complete blood ex-
amination, it is rather difficult to draw up the blood in the pipettes,

2OO MICROMETRY AND BLOOD PREPARATIONS

dilute it, and then get material for fresh blood preparations and films
without undue squeezing, which is to be avoided. Of course, fresh
punctures can be made. Ordinarily, complete blood examinations are
not called for. It is only a white count or a differential count or
an examination for malaria that is required.

As a practical point it is very rare that a red count is indicated. There is one
point not sufficiently recognized by physicians and that is that a routine blood
examination is not apt to be as carefully conducted as one calling for a specific fea-
ture. Without disparaging the necessity of routine examinations of urine as well
as blood it is a fact that the internist who knows what he wants gets better results
from the laboratory man.

HEMOGLOBIN ESTIMATION

The most accurate instrument for this purpose is the Miescher
modification of the v. Fleischl haemoglobinometer.

The magenta-stained glass wedge for comparison with the diluted blood is similar
in each instrument, but by the use of a diluting pipette accurate dilutions are possible
in the Miescher. There are two cells provided — one 12 mm. high, the other 15 mm.
the idea of this being to enable one to make separate comparisons and to select
the central part of the glass-wedge scale, where comparison is more accurate than
at the ends. As these cells contain columns of diluted blood proportionately as 5
to 4, we should have similar readings when we multiply the reading on the scale
with the 15 mm. cell by ££.

The mixing pipette is graduated with the marks J^, %j and K — the first giving a
dilution of i to 400 (when the diluent, a 0.1% soda solution, is drawn up to the mark
above the bulb) the second of i to 300 and the last of i to 200.

Artificial light preferably from a candle is necessary. There is a table accom-
panying each instrument which shows the value for that particular instrument in
milligrams per liter of haemoglobin for any reading obtained on the scale. The nor-
mal amount of haemoglobin in the blood is usually given as 13 to 14 grams per looc.c.
blood. For the first two weeks after birth the amount is much higher, 16 to 20 grams
per 100 c.c. After this time it begins to drop so that a child from five or six
months to twelve or fifteen years old only has about n grams in 100 c.c. blood.

Sahli found variations in normal individuals of from 13.7 grams to 17.3 grams per
100 c.c. of blood.

The apparatus is expensive, requires considerable time and care in the making of
estimations, and is exclusively an instrument for a well-equipped laboratory.

Sahli's Haemometer. — A simple and apparently very scientific
instrument which has been recently introduced is the Sahli modifica-
tion of the Gower haemoglobinometer. Instead of the tinted glass, or
gelatin colored with picrocarmine to resemble a definite blood dilution,
Sahli uses as a standard the same coloring matter as is present in the

HAEMOGLOBIN ESTIMATIONS

201

tube containing the blood. By acting on blood with 10 times its
volume of N/io HC1, haematin hydrochlorate is produced, which gives
a brownish-yellow color. In the standard tube, which is sealed, a
dilution representing i% of normal blood is used.

To apply this test, pour in N/io HC1 to the mark 10 on the scale of the graduated
tube. Add to this 20 cu. mm. of the blood to be examined, drawn up by the capil-
lary pipette provided. So soon as the mixture as-
sumes a clear bright dark brown color, add water
drop by drop until the color of the tubes matches.
The reading of the height of the aqueous dilution
on the scale gives the Hb. reading. The tubes are
encased in a vulcanite frame with rectangular aper-
tures. This gives the same optical impression as
would planoparallel glass sides.

The most accurate readings are obtained with arti-
ficial light in a dark room but almost as satisfactory
comparisons can be obtained with natural light from
a window. It is advisable to turn the ruled side
around so that one may match colors without being
influenced in his determination by the scale.

The apparatus must be kept in a dark place as
strong light will change the color of the standard
tube. It is recommended that the N/io HC1 be
preserved with chloroform.

Tallquist's Haemoglobin Scale. — This is
a small book of specially prepared filter-
paper with a color-scale plate of 10 shades
of blood colors. These are so tinted as to
match blood taken up on a piece of the filter-
paper and are graded from 10 to 100. So
soon as the blood on the filter-paper has lost
its humid gloss, the comparison should be
made.

FIG. 52. — Sahli's haemo-
globinometer. (Greene.)

This may be done by shifting the blood-stained piece of filter-paper suddenly
from one to the other of the holes cut in each shade— the piece of filter-paper being
underneath the color plate; it is better, however, to match the colors with the blood
spot against the scale rather than behind a preparation. Grawitz prefers to cut the
stained spot from the filter-paper and place it directly on the color scale.

At least a square centimeter of the filter-paper should be stained
by the blood. Daylight coming from a window to the rear or at the side
should be used in making the comparison. The error with this method

2O2

MICROMETRY AND BLOOD PREPARATIONS

is probably not over 10% after a little experience.
is not kept in the dark, the tints tend to fade.

If the colored plate

To COUNT BLOOD-CORPUSCLES

The instrument almost universally used is the Thoma-Zeiss haemacy-
tometer. The apparatus consists of two pipettes, one for leukocytes,
graduated to give a dilution of i to 10 or greater; the other for red cells
to give a dilution of a i to 100 or greater. The white pipette has the
mark n above the bulb and the red pipette the mark 101. In addition,
there is a counting chamber.

This consists of a square of glass with a round hole in the center. Occupying the
center of this round hole is a circular disc of glass of less diameter, so that an encirc-

ling channel is left. The square and the circle
of glass are cemented to a heavy glass slide.
The surfaces of each are absolutely level and
highly polished. That of the circular disc is
ruled into squares of varying size and is exactly
Ho rnm. below the level of the surface of the sur-
rounding glass square.

When a polished piano-parallel cover-
glass rests on the shelf, as the outer square
glass is termed, there is a space left be-
tween its under-surface and the ruled disc
of o.i mm. The channel around the disc
is termed the moat or ditch.

The most desirable rulings are those of Tiirck
and of Zappert. In these the entire ruled surface
consists of nine large squares, each i mm. square.
These are subdivided, and in the central large
small squares used for averaging the red cells.

FIG. 5 3 . —Thomas-Zeiss
blood counter showing pipette,
counting chamber, and ruled
field. (Greene.}

the

square are to be found
These small squares are Ho rnm. square and are arranged in nine groups of 16
small squares by bordering triple-ruled lines. As the unit in blood counting is
the cubic millimeter, if one counted all the white cells lying within one of the
large squares (i mm. square), he would have only counted the cells in a layer
one-tenth of the required depth, so that it would be necessary to multiply the
number obtained by 10. This product, multiplied by the dilution of the blood,
would give the number of white cells in a cubic millimeter of undiluted blood.
Some workers prefer the Biirker hamacytometer. In this there are two ruled
wedge-shaped pieces of glass, separated at their bases, which take the place of
the ruled disc of the Thoma apparatus. Two oblong pieces of glass are on either
side of the ruled wedges and are o.i mm. higher, thus taking the place of the shelf.
Clamps fix a cover-glass on these shelves giving a space Ho mm. over the ruled sur-

RED CELL COUNTS

203

faces. The blood is run in by capillarity from the mixing pipette. I gave up this
type of counter because the clamps made manipulation awkward and because the
usual apparatus is most satisfactory.

Red Cell Counts. — To make a red count: Having a fairly large drop of blood, ap-
ply the tip of the 101 pipette to it and, holding the pipette horizontally, carefully
and slowly draw up with suction on the rubber tube a column of blood to exactly
0.5 or i. The variation of ^5 inch from the mark would make a difference of al-
most 3%. If the column goes above 0.5, it can be gently tapped down on a piece
of filter-paper until the 0.5 line is cut. Now insert the tip of the pipette into some
diluting fluid and, revolving the pipette on its long axis while filling it by suction,
you continue until the mark 101 is reached. A variation of ^5 inch at this mark
would only give an error of about one- thirtieth of i%. After mixing thoroughly by
shaking for one or two minutes, the fluid in the pipette below the bulb is expelled
(this, of course, is only diluting fluid). A drop of the diluted blood of a size just
sufficient to cover the disc when the cover-glass is adjusted, is then deposited on the
disc and the cover-glass applied by a sort of sliding movement, best obtained by using
a forceps in one hand assisted by the thumb and index-finger of the other.

Among diluting fluids Toisson's is probably the best:

Sodium chloride i gram

Sodium sulphate 8 grams

Glycerine 30 c.c.

Distilled water 160 c.c.

Dissolve the sodium chloride and the sodium sulphate in the glycerine water and
add sufficient methyl or gentian violet to give a rich violet tint.

Hayem's solution is very satisfactory and is preferred by many workers. It has
the following composition: Corrosive sublimate 0.5 gram, Sod. chloride i gram,
Sod. sulphate 5 gram, 200 c.c. of distilled water.

A 2^% solution of potassium bichromate makes a very satisfactory diluting
fluid in the counting of red cells.

A salt solution of about i% strength, tinged with about i drop of a saturated
alcoholic solution of gentian violet to about 50 c.c., is a good substitute, or the salt
solution alone will answer when no white count is to be made at the same time as the
red one.

It is important to work quickly in adjusting the cover-glass, or there will be cells
settling in the center of the drop from a greater depth than the one which the apposi-
tion of the cover-glass makes (Ko mm- deep).

A good preparation should show:

1. Presence of Newton's rings.

2. Absence of air bubbles.

3. Entire surface of ruled disc covered.

4. Equal distribution of cells.

Before counting, about five minutes should be allowed for the set-
tling of the cells.
It will be remembered that the small squares are ^o mm. square. The

204 MICROMETRY AND BLOOD PREPARATIONS

depth of fluid from upper surface of shelf to lower surface of cover-glass is Mo mm.
Hence each space embraced by the small square and the depth of fluid is Mo 00
of the unit used in estimating number of corpuscles in blood, or i cu. mm.
(Mo X Mo X Mo = Mooo)- Count 100 of the small squares (this enables one
to use decimals). There are nine squares between triple-ruled lines, each containing
1 6 small squares. Count the number of corpuscles in the 16 small squares
contained in upper left-hand triple-ruled square. Put down this count. Next
count corpuscles in the adjoining 1 6 squares. Put down this count. Then in
third 1 6 squares. Put down the number. Now move down to next row of
three triple-ruled squares. Count the number of corpuscles in each of the three
i6-square spaces and set down the numbers for addition. We have now counted
96 small squares (6 X 16). Count at any place four additional small squares and
add number of blood-cells contained therein to those in the 96 small squares
already counted. Divide the sum by 100 or simply point off two decimals.
This gives the average for each small square. Multiply this by the dilution and then
(as the small square is only Mooo cu. mm.) by 4000. This will give the number of
corpuscles in i cu. mm. Example: 100 small squares contained 655 red cells.
Pointing off, 6.55 equals average number of red cells per small square. Multiply
by dilution (200) and then by 4000 (the small square is 4000 times smaller than the
unit: i cu. mm.) — 6.55 X 200 = 1310 X 4000 = 5,240,000.

At least ioo small squares, and preferably 200 should be counted.
If the blood appears normal, one may simply count the number of red
cells in five of the 16 small square spaces (80 small squares). Having
added the numbers and multiplying by 10,000, you obtain the number
of cells in i cu. mm. (Eighty small squares is J-^Q of the unit of i
cu. mm., or 4000 small squares. The blood dilution being i to 200, we
have 50 X 200 X number of cells in 80 small squares.)

In counting, count corpuscles lying on the lines above and to the right. Do not
count those lying on lines below and to the left.

In the small squares count only corpuscles lying in the space or cutting the upper
line. This prevents counting the same cell twice.

To Count White Cells. — Draw up the blood in the white pipette to
the mark 0.5. Then, still holding the pipette as near the horizontal as
possible, because the column of blood tends to fall down in the larger
bore, draw up by suction a diluting fluid which will disintegrate the red
cells without injuring the whites.

The best fluid is 0.5% of glacial acetic acid in water. This makes the white cells
stand out as highly refractile bodies. Some prefer to tinge the fluid with gentian
violet. The 0.5 mark is preferred because it takes a very large drop of blood to
fill the tube up to the i mark and if there is much of a leukocytosis a i to 10 dilution
is not sufficient. In leukaemic blood it is better to use the red pipette with the
0.5% acetic acid solution.

The blood having been drawn up to 0.5, we have a dilution of i to 20.

LEUCOCYTE COUNTS 205

Making a preparation, exactly as was done in the case of the red count, we count
all of the white cells in one of the large squares (i sq. mm.). The cross ruling greatly
facilitates this. Note the number. Then count a second and a third large square.
Strike an average for the large squares counted and multiply this by 10, as the depth
of the fluid gives a content equal to only ^ 0 cu. mm. Then multiply by the
dilution. Example: First large square 50; second large square 70; third large
square 60. Average 60. Then 60 X 10 X 20 = 12,000, the number of leukocytes in
i cu. mm. of blood. The count may be made with a low power (%-inch objec-
tive) as the leukocytes stand out like pearls. It is more accurate, however, to use a
higher power, so that pieces of foreign material may be recognized and not enumer-
ated as white cells. If one will accustom himself to comparing the distribution of
the leukocytes in a well-made stained dried blood film, prepared according to Ehrlich's
cover-glass method, with that in a haemacytometer preparation, he can readily
acquire an experience which will enable him to determine with considerable accuracy
the degree of leukocytosis by the examination of a stained cover-glass preparation
alone. Furthermore, one can identify the leukocytes in a Giemsa-stained smear
with the %-inch objective. This is specially true of the large mononuclears and
transitionals whose increase has such significance in the tropics.

Special diaphragms for the ocular, with a square opening, which just covers one
of the large squares of the haemacytometer (400 small squares) may be purchased
or one may cut a square hole from a round piece of stiff paper which rests on the ocular
micrometer supports. This is easily measured by noting the number of spaces on
the ocular micrometer which equals one side of the square. With such an opening
one can count the leukocytes in unruled areas equal to a large square, when the ruling
is less convenient than is present in the Ttirck ruling.

Combined Red and White Counts. — In the absence of a white pipette or when
it is desired to make a white count with the same preparation as is used for the red
one, especially if the ruling is of the old style (only central ruling and not in nine
large squares as with Zappert and Tiirck), it is advisable to make use of the method
of counting by fields. With a Leitz No. 4 ocular and a No. 6 objective, with a tube
length of 120 mm., it will be observed that the field so obtained has a diameter
of eight small squares. Now, remembering that the area of a circle equals the square
of the radius multiplied by TT, or 3.1416, we have the following calculation: The
diameter being eight small squares, the radius would be four small squares. Squar-
ing the radius, we have 16. This multiplied by 3.1416 gives us 50. This
means that every field, with the microscope adjusted as stated, contains 50 of
the small squares, or Ho of the unit of i cu. mm. of the diluted blood.

By keeping a single red cell in view while moving the mechanical stage from right
to left or from above downward, we know that a new field of 50 small squares is
brought into view when the circumference of the field cuts this individual cell.
Example: As 2000 small squares would ordinarily be a sufl&cient number to count
for a white count, this would require us to count the number of leukocytes in 40
of the designated microscopic fields (this, of course, is only one-half the unit, hence
we should multiply by 2). Counted 40 fields and noted 50 white cells. 50 X 2
= 100 X 200 (the dilution in red pipette) = 20,000. Consequently 20,000 would
represent the number of leukocytes in i cu. mm. of the blood examined.

Cleaning Apparatus.— After making a blood count, the haemacytometer slide
should be cleaned with soap and water and then rubbed dry, preferably with an old

206 MICROMETRY AND BLOOD PREPARATIONS

piece of linen. As the accuracy of the counting chamber depends upon the integrity
of the cement, any reagent such as alcohol, xylol, etc., and, in particular, heat, will
ruin the instrument. The pipettes should be cleaned by inserting the ends into the
tube from a vacuum pump, as a Chapman pump. First draw water or i% sod.
carbonate solution through the pipette, then alcohol, then ether, and finally allow
air to pass through to dry the interior. If the interior is stained, use i% HC1 in
alcohol. If a vacuum pump is not at hand, a bicycle pump or suction by mouth
will answer.

PREPARATIONS FOR THE STUDY OF FRESH BLOOD

Many authorities prefer a fresh-blood specimen to a stained dried
smear in the study of parasites of the blood. In malaria in particular
there is so much information as to species to be obtained from a fresh
specimen that the employment of this method should never be neglected.
While waiting for the film to stain one has five or six minutes which
could not be better spent than in examining the fresh specimen which
only requires a moment to make.

Manson's Method.— Have a perfectly clean cover-glass and slide. Touch the
apex of the exuding drop of blood with the cover-glass and drop it on the center of
the slide. The blood flows out in a film which exhibits an "empty zone" in the
center. Surrounding this we have the "zone of scattered corpuscles," next the
"single layer zone" and the "zone of rouleaux" at the periphery. It is well to ring
the preparation with vaseline. When desiring to demonstrate the flagellated bodies
in malaria, it is well to breathe on the cover-glass just prior to touching the drop of
blood.

The Method of Ross is very easy of application and gives most satisfactory
preparations. Take a perfectly clean slide, and make a vaseline ring or square of
the size of the cover-glass. Then, having taken up the blood on the cover-glass,
drop it so that its margin rests on the vaseline ring. Gently pressing down the cover-
glass on the vaseline makes beautiful preparations which keep for a very long time.
If it is desired to study the action of stains on living cells, this method is also appli-
cable. A very practical way to do this is to tinge 0.85% salt solution containing i%
sodium citrate (the same as is used in opsonic work) with methylene azur, gentian
violet, or methyl green. With a Wright bulb pipette, take up i part of blood,
then i part of tinted salt solution. Mix them quickly on a slide and then deposit
a small drop of the mixture in the center of the vaseline ring and immediately apply
a cover-glass and press down the margins as before. This method will be found of
great practical value.

A METHOD FOR MAKING DIFFERENTIAL LEUKOCYTE COUNT IN SAME
PREPARATION AS FOR WHITE COUNT

Employ the same technic as in making the ordinary white count
but using as a diluting fluid a ij^ or 2% formalin solution to which has

DIFFERENTIAL COUNTS 207

been added i drop of Giemsa's stain for each c.c. just before making
the blood examination.

The best results are obtained when the mixing in the pipette bulb is done im-
mediately after taking up the blood and diluent. Recently I have found it necessary
to add enough N/i NaOH to the commercial formalin to bring it to about +0.75.
To do this add to it a few drops of phenolphthalein as an indicator and continue
to add a dilute sodium hydrate or sodium carbonate solution until a pink color
just developes at room temperature. This corresponds to about +0.75 with boiling
titration. The acidity of commercial "Formalins" varies greatly. Of this +0.75
formalin I use i^% in a %% glycerine solution instead of water.

The usual technic in making the haemocytometer preparation is employed using
a Tiirck ruling. Count the leukocytes in the three upper or lower i sq. mm. squares,
divide by 3 to obtain an average per sq. mm., multiply by 10 for the content of a
cubic millimeter and then by 20 for the dilution. (Blood to 0.5; diluent to n.)
This can be done mentally and requires no calculation on paper. Having counted
the leukocytes, again go over the same portion of the ruled surface and count the
polymorphonuclears and estimate the percentage of these to the total leukocytes.
The majority of disrupted cells in a dry-stained preparation are transitionals hence
the percentage of polymorphonuclears by this method is lower.

It is unnecessary in such a count to have an assistant; of course, in making a
complete differential count it is preferable to have some one tabulate or laboriously
to do this one's self.

The red cells are practically diaphanous and not disintegrated as
when acetic acid is used as a diluent, consequently it is easy to make
out the particular red cell as to size, etc., containing a malarial parasite.

The best results are obtained with a %-inch objective. Higher powers are of
course impracticable by reason of the thickness of the cover-glass of the haemocyto-
meter.

The following are the appearances of the various leukocytes.

Eosinophiles. — In these the bilobed nucleus stains rather faintly and the color
is greenish blue. The eosinophile granules show easily as coarse, brickdust-red
colored particles.

Polymorphonuclears. — The nucleus stains a deep, rich, pure violet but less in-
tense than that of the small lymphocyte. The shape of the nucleus is typically
three or four lobed but even when of the horseshoe shape of a transitional nucleus
is easily recognizable by the intensity of the violet staining. That which makes the
polymorphonuclears very easy of differentiation is the distinctness of the cell out-
lines produced by the fine yellowish granulations in the cytoplasm.

Small Lymphocytes. — The nucleus is perfectly round and stains a deep violet.
It is almost impossible to make out any cytoplasmic fringe.

Large Lymphocytes. — The nucleus here is round and of a lighter violet than that
of the small lymphocyte. The cytoplasm is blue, nongranular, and sharply defined
from the nucleus.

Large Mononuclears. — These show a washed-out, slate-colored nucleus which
blends with the gray slate-blue staining of the cytoplasm so that there is an in-
defmiteness of outline in the more or less irregularly contoured nucleus.

208 MICROMETRY AND BLOOD PREPARATIONS

Transitionals. — These show the same characteristics as the large mononuclears,
but with a more faintly stained and more indented nucleus. The large mono-
nuclears and transitionals stand out as slate-colored cells. When very much degen-
erated these cells have a greenish hue.

Mast Cells. — The granulations show as a rich maroon or reddish-violet color

The young ring forms of malaria show as violet-blue areas in the red cells. When
half-grown or approaching the merocyte stage, the containing red cell takes on a
faint pink coloration, thereby differentiating it from the noninfected red cells. At
the same time the parasite is extruded and has the appearance of a violet-blue body
projecting from the margin of the red cell. It is as if a blue body were budding from
a pink one. Crescents stand out very distinctly.

It is an easy matter with this method to count the number of trypanosomes or
malarial crescents in a cubic millimeter of blood.

PREPARATIONS AND STAINING OF DRIED FILMS

When preparations are desired for a differential count, Ehrlich's
method of making films is to be preferred, as the different types of
leukocytes are more evenly distributed. In making smears by spread-
ing, there is a tendency for the polymorphonuclears to be concentrated
at the margin while lymphocytes remain in the central part of the film.

In Ehrlich's method we have perfectly clean dry cover-slips. Take up a small
drop of blood without touching the surface of the ear or finger. Drop this cover-
glass immediately on a second one and as soon as the blood runs out in a film, draw
the two cover-slips apart in a plane parallel to the cover-glasses. Slide them apart.
Ehrlich uses forceps to hold the cover-glasses to avoid moisture from the fingers,
but I find I can work more quickly and satisfactorily with the fingers alone. In
making malarial smears it is better to wash the finger or ear with soap and water
to get rid of all grease and dirt. Then dry thoroughly before puncturing. Alcohol
is not so efficient.

Slides and spreaders should be absolutely clean and grease free. Scrubbing with
soap and water, thorough rinsing and drying, then subjecting the slide to the flame
to make it grease free is satisfactory.

Of the various methods of spreading films on slides there is none
equal to that described by Daniels. In this the drop of blood is drawn
along and not pushed along. The films are even, can be made of any
desired thickness by changing the angle of the drawing slide, and there
is little liability of crushing pathological cells. Take a small drop of
blood on the end of a clean slide. Touch a second slide about %
inch from end with the drop and as soon as the blood runs out along
the line of the slide end, slide it at an angle of 45° to the other end of the
horizontal slide. The blood is pulled or drawn behind the advancing

BLOOD SMEARS

2OO

edge of the advancing slide. An angle less than 45° makes a thinner
film; one greater, a thicker film.

Instead of a slide a square cover-glass may be used and if the edge be smooth it
makes a more satisfactory spreader than the slide.

Instead of the Daniels method some prefer to take up the drop of blood on the
slide on which the smear is to be made, about Y2 inch from the end. Then apply
the spreader slide and so soon as the drop runs along the end of the spreader slide
proceed as above described.

FIG. 54. — Blood technic. i, 2, 3, Method for making blood smear on slide; 4,
U tube for resting slides while staining; 5, slide showing grease pencil marking,
marking prevents stain from overflowing; 6, method for drawing apart cover-glasses
in making blood smear.

Of the various methods of making smears by means of cigarette paper, rubber
tissue, needles, etc., the best seems to be to take a piece of capillary glass tubing and
use this instead of a needle in making the film. There is one advantage about the
strip of cigarette paper touched to the drop of blood and drawn out along the slide
or cover-glass, and that is that it is almost impossible not to make a working prepara-
tion by this method.

In the making of smears the chief points are to make the smears as
soon after taking the blood as possible and to have slides and cover-
glasses scrupulously clean. It is well to flame all slides and cover-
14

210 MICROMETRY AND BLOOD PREPARATIONS

glasses which are to be used for blood-work. This is the best method
of getting rid of grease.

Thick Film Methods. — Such methods are of the greatest practical
value in diagnosis of malarial parasites when in very small numbers
in the peripheral circulation. They are also of great value in finding
trypanosomes, relapsing fever spirochaetes and filarial embryos in the
blood. Ruge's method so brings out the polymorphonuclears that
such a technic can be used for opsonic index.

Method of Ross. — In this about one-half of a drop of blood is smeared out over a
surface about equal to that of a square cover-glass and allowed to dry. It is then
flooded with a 0.1% aqueous solution of eosin for about fifteen minutes. The prepa-
ration is then gently washed with water and then treated with a polychrome
methylene-blue solution. After a few seconds this is carefully washed off and the
preparation dried and examined.

Method of James. — -James smears out an ordinary drop of blood so that it makes a
circular smear about % inch in diameter. This may be easily accomplished with a
spatulate wooden toothpick. When dry, treat the blood smear with alcohol contain-
ing HC1. (Alcohol 50 c.c. HC1. 10 drops) until the haemoglobin is dissolved out.
Then wash thoroughly in water for five or ten minutes. Allow to dry and then
stain as ordinarily with the Wright or Giemsa stain.

Ruge's Method. — The best thick film method is that of Ruge. After the blood
has dried well, gently move the slide about in a glass containing a 2% solution of
formalin to which has been added i% of glacial acetic acid. After laking is com-
plete, as shown by disappearance of brown color, treat the slide in the same way in a
glass of tap water to remove all traces of acid. Next wash very gently in distilled
water and stain with dilute Giemsa (i drop to i c.c.) for twenty to thirty minutes.
Wash in water and allow to dry without heat or blotting paper.

Some workers prefer to stain the dried thick smear for one hour in a jar containing
dilute Giemsa stain (i to 40) without previous fixation or dehaemoglobinization.

At present, I make my thick films by taking up a moderately large
loopful from the exuding drop of the puncture wound.

This is deposited at one end of the slide and from it three or four more
daubs are made in succession toward the other end of the slide.
These daubs are quickly smeared out before coagulation takes place
in the first daub.

With all thick film methods it is extremely important to have thorough drying
of the smear before dehaemoglobinizing or staining. This ordinarily requires one or
two hours in the air or twenty to thirty minutes in the incubator. It is particularly
important in working with such smears, although holding for ordinary smears, to
protect them from flies, ants, etc., as such insects will eat up the smear in a few
minutes if left exposed.

Fixation of Film. — In Wright's, Leishman's, and other similar stains the methyl-
alcohol solvent causes the fixation. In staining with Giemsa's stain, Ehrlich's

STAINING BLOOD-FILMS 211

triacid, hasmatoxylin and eosin, Smith's formol fuchsin, and with thionin, separate
fixation is necessary. For Giemsa and thionin, either absolute alcohol (ten to fifteen
minutes), or methyl alcohol (two to five minutes) answer well.

Formalin vapor, for five to ten seconds, is also used for fixation. For Ehrlich's
triacid, haematoxylin and eosin and formol fuchsin, heat gives the best results.
The best method is to place the films in an oven provided with a thermometer.
Raise the temperature of the oven to i35°C. and then remove the burner. After
the oven has cooled, take out the fixed slides or slips.

Some prefer to place a crystal of urea on the slide, then hold it over the flame
until the urea melts. This shows that a temperature between 130° and i35°C. has
been reached.

One of the handiest methods is to drop a few drops of 95% alcohol on the slide
or cover-glass. Allow this to flow over the entire surface; then get rid of the exeess
of alcohol by touching the edge to a piece of filter-paper for a second or two. Then
light the remaining alcohol film from the flame and allow the burning alcohol to
burn itself out. A chemical fixation which gives good fixation for haematoxylin
and triacid stains (not equal to heat) is a modification of Zenker's fluid (Whitney).
To Miiller's fluid, which is potassium bichromate 2 grams, sodium sulphate i gram,
and water 100 c.c., add 5 grams of bichloride of mercury and 5 c.c. of nitric acid
(C. P.). Fixation is obtained in five seconds.

When using corrosive sublimate fixation one should after thorough washing in
water treat the film with Gram's iodine solution for about two minutes and then wash
with 70% alcohol until the yellow color of the film disappears. Methyl alcohol for
two minutes is satisfactory. (See Staining of Protozoa.)

Staining Blood-films. — As separate staining with eosin and methyl-
ene blue rarely gives good preparations and as the modifications of the
Romanowsky stain recommended are easy to make and employ, and
give much greater information, the separate method of staining is not
recommended. The most satisfactory single stain is thionin.

Rees' Thionin Solution— Take of thionin 1.5 grams, alcohol 10 c.c., aqueous solution
of carbolic acid (5%) 100 c.c. Keep this as a stock solution. It should be at least
two weeks old before using. For use, filter off 5 c.c. and make up to 20 c.c. with
water.

1. Fix films (a) by heat, (b) by alcohol and ether, or (c) preferably by i%
formalin in 95% alcohol for one minute.

2. Stain for from ten to twenty minutes. Wash and mount. Malarial parasites
are stained purplish; nuclei of leukocytes, blue; red ceUs, faint greenish blue.

Ehrlich's Triacid or Triple Stain.— There are required:

1. Sat. aq. sol. orange G. (Dissolve 3 grams in 50 c.c. water.)

2. Sat. aq. sol. acid fuchsin. (Dissolve 10 grams in 50 c.c. water.)

3. Sat. aq. sol. methyl green. (Dissolve 10 grams in 50 c.c. water.)

These three solutions may be kept as stock solutions. They keep well in the dark.
To make the stain, add 9 c.c. of No. 2 (acid fuchsin) to 18 c.c. of No. i (orange G.).
After they are mixed thoroughly, add 20 c.c. of No. 3 (methyl green). Then after
the first three ingredients are well mixed, add 5 c.c. of glycerine. Mix, then add 15

212 MICROMETRY AND BLOOD PREPARATIONS

c.c. of alcohol; again mix, and finally add 30 c.c. of distilled water. Keep the
mixed stain about one week before using. The best fixatives are heat and Whitney's
modified Zenker. To use, stain films from two to five minutes; then wash and
mount. The triacid stain is a good tissue stain. The objections to the triacid
stain are that it does not stain malarial parasites or mast cells and that failure to
obtain good results is of frequent occurrence.

Wright's Method. — The stain is made by adding i gram of methylene blue (Grub-
ler) to 100 c.c. of a %% solution of sodium bicarbonate in water. This mixture is
heated for one hour in an Arnold sterilizer. The flask containing the alkaline methyl-
ene-blue solution should be of such size and shape that the depth of the fluid does
not exceed 2% inches. When cool, add to the methylene-blue solution 500 c.c. of
a i to 1000 eosin solution (yellow eosin, water soluble). Add the eosin solution
slowly, stirring constantly until the blue color is lost and the mixture becomes
purple with a yellow metallic luster on the surface, and there is formed a finely
granular black precipitate. Collect this precipitate on a filter-paper and when
thoroughly dry (dry in the incubator at 38°C.) dissolve 0.3 gram in 100 c.c. of pure
methyl alcohol (acetone free). Wright lately has recommended using o.i in 60 c.c.
methyl alcohol. This constitutes the stock solution. For use filter off 20 c.c. and
add to the filtrate 5 c.c. of methyl alcohol.

A modification by Batch is very satisfactory. In this method instead of poly-
chroming the methylene blue with sodium bicarbonate and heat, the method of
Borrel is used. Dissolve i gram of methylene blue in 100 c.c. of distilled water.
Next dissolve 0.5 gram of silver nitrate in 50 c.c. of distilled water. To the silver
solution add a 2 to 5% caustic soda solution until the silver oxide is completely pre-
cipitated. Wash the precipitated silver oxide several times with distilled water.
This is best accomplished by pouring the wash-water on the heavy black precipitate
in the flask, agitating, then decanting and again pouring on water. After removing
all excess of alkali by repeated washings, add the methylene-blue solution to the
precipitated silver oxide in the flask. Allow to stand about ten days, occasionally
shaking until a purplish color develops. The process may be hastened in an incu-
bator. When polychroming is complete, filter off and add to the filtrate the i to
1000 eosin solution and proceed exactly as with Wright's stain.

In Leishman's method the polychroming is accomplished by adding i gram of
methylene blue to 100 c.c. of a V^% solution of sodium carbonate. This is kept at
65°C. for twelve hours and allowed to stand at room temperature for ten days
before the eosin solution is added. The succeeding steps are as for Wright's stain.

In all Romanowsky methods distilled water should be used. If not
obtainable, the best substitute is rain-water collected in the open and
not from a roof.

If the yellow color in water in a test-tube to which has been added a small pinch of
haematoxylin does not change to blue in from one to five minutes it is too acid and
should be treated with a i % sod. carb. sol. until it does show blue. Alkaline waters
are less easy to correct.

Method of staining:

1. Make films and air dry.

2. Cover dry film preparation with the methyl-alcohol stain for one minute (to
fix).

GIEMSA'S STAIN 213

3. Add water to the stain on the cover-glass or slide, drop by drop, until a yellow
metallic scum begins to form. It is advisable to add the drops of water rapidly in
order to eliminate precipitates on the stained film. Practically, we may add i drop of
water for every drop of stain used.

4. Wash thoroughly in water until the film has a pinkish tint.

5. Dry with filter-paper and mount. The stained preparation is less apt to show
foreign material and damage if one allows the film to dry without blotting. In a
moist atmosphere one may dry the film high over the flame but any near contact
with the heat of the flame is injurious.

Red cells are stained orange to pink; nuclei, shades of violet; eosino-
phile granules, red; neutrophile granules, yellow to lilac; blood platelets,
purplish; malarial parasites, blue; chromatin, metallic red to rose pink.

The bottle in which the methyl alcohol solution is kept must be tightly stoppered
with a cork stopper and kept in the dark. Any evaporation of the alcohol interferes
with proper fixation of the blood film and the light affects the delicate stain.

Giemsa's Modification of the Romanowsky Method. — This is one of
the most perfect of the modifications. The objection is that greater
time in staining films is required than with the Wright or Leishman
method and the stain is very expensive.

Take of Azur II eosin 0.3 gram. Azur II 0.08 gram.

Dissolve this amount of dry powder in 25 c.c. of pure anhydrous glycerine at 6o°C.
Then add 25 c.c. of methyl-alcohol at the same temperature. Allow the glycerine
methyl-alcohol solution to stand over night and then filter. This is the stock stain.
To use: Dilute i c.c. with 10 to 15 c.c. of distilled water. If i to 1000 potassium
carbonate solution is used instead of water it stains more deeply.

The alkaline diluent is used to obtain the course stippling in malig-
nant tertian (Maurer's clefts). Having fixed the smear with methyl
alcohol for one to five minutes, pour on the diluted stain, and after
fifteen to thirty minutes wash off and continue washing with distilled
water until the film has a slight pink tinge. For Treponema pallidum
stain from two to twelve hours.

While the Romanowsky methods are more satisfactory for differential counts and
for the demonstration of the malarial parasites, and especially for differentiating
species, yet by reason of the liability to deterioration in the tropics of methylene
blue the haematoxylin methods may be preferable. Many workers in blood-work
and cytodiagnosis prefer the haematoxylin.

1. Fix the film either by heat, with methyl alcohol for two minutes or with Whit-
ney's fixative. Heat is to be preferred.

2. Stain with Meyer's hemalum or Delafield's haematoxylin for from five to
fifteen minutes according to the stain. Frequently three minutes will be found
sufficient. To make the hemalum, dissolve 0.5 gram of haematin in 25 c.c. of 95%

214 MICROMETRY AND BLOOD PREPARATIONS

,.

tis-

alcohol. Next dissolve 25 grams of ammonia alum in 500 c.c. of distilled wat
Mix the two solutions and allow to ripen for a few days. The stain should be satis-
factory in two or three days.

To make Dela field's haematoxylin, dissolve i gram of haematoxylin crystals in
6 c.c. of 95% alcohol. Add this to 100 c.c. of saturated aqueous solution of ammonia
alum. After exposure to light for a week, the color changes to a deep blue purple.
Add to this ripened stain 25 c.c. of glycerine and 25 c.c. of methyl-alcohol and, after
it has stood for about two days, filter. The stain should be filtered from time to time
as a sediment forms. This makes a stock solution which should be diluted 10 to 15
times with water when staining.

Mink's Modification of Unna's Haematoxylin

Haematoxylin i gram

Alum 8 grams.

Sulphur (sublimed) i gram.

Glycerine 30 c.c.

Alcohol 50 c.c.

Water 100 c.c.

Dissolve the haematoxylin in the glycerine in a mortar. Dissolve the alum in the
water and add it to the glycerine hsematoxylin in the mortar. Then add the sulphur
and the alcohol. The solution ripens in about three to four days. Allow the sedi-
ment to remain in the bottom of the bottle containing the stain and filter off small
quantities as needed.

3. Wash for two to five minutes in tap water to develop the haematoxylin color.

4. Stain either with a i to 1000 aqueous solution of eosin or with a one-half of i %
eosin solution in 70% alcohol. The eosin staining only requires fifteen to thirty
seconds.

5. Wash and examine.

IODOPH3LIA

This reaction is supposed to be due to the presence of glycogen,
especially in the polymorphonuclears, in suppurative conditions.

It has been stated that a differentiation between the joint involvement in gonor-
rhceal infection and in articular rheumatism may be made from iodophilia being
present in the gonococcus infection.

Make blood-smears on cover-glasses as usual, and after they dry, but without
fixation, mount them in a drop of the following solution:

Iodine i part.

Potassium iodide 3 parts.

Gum arabic 50 parts.

Water 100 parts.

Small brown masses in the polymorphonuclears indicate a positive iodophilia.
Viscosity of the Blood. — This is estimated by observing the relative height to which
blood rises in capillary tubes as compared with water, and normally varies from three

COAGULATION RATE 215

to five. The higher the haemoglobin content the greater the viscosity. Viscosity is
high in arterio-sclerosis and diabetic coma, low in the anaemias of nephritis.

Coagulation Rate of Blood. — This determination is of value in
connection with operations on jaundiced patients.

Wright's coagulometer is a standard instrument but is cumbersome.

A simple method of determining the rate is to take a piece of capillary glass tubing
and hold it downward from the puncture to let it fill for 3 or 4 inches. Then at inter-
vals of thirty seconds scratch with a file the capillary tubing at short distances and
break off between the fingers. When coagulation has taken place a long worm-like
coagulum is obtained. Normally coagulation occurs in about three to four minutes,
when the temperature is that of the hand in which the tubes are conveniently held.
Rudolf recommends placing the tubes in metal tube containers in a Thermos bottle
at 2o°C. He gives the normal coagulation rate for this temperature as eight minutes,
while at a temperature below this the period is lengthened. Age and sex do not
influence the rate. Sabrazes, the originator of this method found no appreciable
variation in tubes from 0.8 to 1.2 mm. diameter.

In Barker's test you mix a drop of blood and a drop of distilled water on a slide
and with a capillary tube sealed off at the end stir the mixture every half minute.
So soon as fibrin threads appear you have coagulation.

SPECIFIC GRAVITY OF THE BLOOD

Hammerschlag has a method for the determination of the Hb.
percentage based upon the specific gravity of the blood.

In this method a mixture of benzol and chloroform is made of a specific gravity of
about 1050. A medium size drop of blood is then taken up with a pipette and intro-
duced below the surface of the mixture, carefully avoiding production of bubbles.
Variation in temperature introduces a very appreciable error. If it sinks add more
chloroform from a dropping bottle, if it tends to rise, more benzol. The mixture in
which the drop of blood tends to remain stationary, near the top of the mixed benzol
and chloroform, has the same specific gravity as that of the blood. This is deter-
mined by an accurately graduated hydrometer. The normal average specific
gravity for men is 1059, for women 1056. A table, giving the Hb. percentage cor-
responding to the specific gravity, accompanies the outfit.

Eykmann controls the specific gravity of the drop of blood by adding a drop c
salt solution made to have a similar specific gravity.

The specific gravity is reduced in all anaemias, especially chlorosis; in nephritis witl
osdema as well as in most cachetic states.

In these latter the Hb. percentage may be normal.

Specific Gravity in Cholera.— To determine the necessity for intraven-
ous infusion in cholera Rogers has recently recommended the employ-
ment of small bottles containing aqueous solution of glycerine with
specific gravities varying from 1048^0 1070, increasing the spec
gravity in each successive bottle by 2°.

2l6 MICROMETRY AND BLOOD PREPARATIONS

An accurate urinometer will suffice to determine the specific gravity. Drops of
blood from the cholera patient are deposited at the center of the surface of the fluid
in the bottles from a capillary pipette. If the specific gravity of the blood is 1062
at least a liter of saline or sodium bicarbonate solution is needed. If 1066 at least
2 liters. Formerly he estimated the indications by blood-pressure considering a
pressure of 80 in Europeans or of 70 in natives as indicating intravenous injections.

TESTS FOR AGGLUTINATION AND HAEMOLYSIS OF THE RED CELLS
(TRANSFUSION)

In the selection of a donor for blood for transfusion it is always
necessary to try his red cells against the serum of the recipient as well
as the patient's red cells against the serum of the donor, in order to
prove the absence of haemolyzing or agglutinating bodies.

Certain persons have isohaemolysins in their blood which dissolve the red cells of
other persons and in paroxysmal haemoglobinuria autohaemolysins may be present
which can destroy the patient's own red cells. This autohaemolysin seems operative
only when a low temperature is followed by a high one. When haemoglobinaemia
exists the liver converts it into bile pigment, causing bilious stools and jaundice. If
one-sixth of the red cells are destroyed hEemoglobinuria results.

Before transfusing carry out the following tests:

From a vein take about i c.c. of blood in a centrifuge tube containing i % of sod.
citrate salt solution; then shift the stopper of the blood system to a dry centrifuge
tube and draw into it about 3 or 4 c.c. of blood. Throw down the citrated blood,
pipette off the supernatant fluid and wash the sediment with normal saline.

Again pipette off the saline after centrifuging and make a 10% emulsion of the red-
cell sediment in normal saline.

Centrifuge the coagulated blood in the other tube and collect the serum which
separates from the clot.

Carry out these procedures for both donor and recipient.

Tests: i. In a small test-tube deposit i drop of the donor's 10% red-cell emulsion
and then add 4 drops of the recipient's serum.

2. Treat similarly i drop of the recipient's red-cell emulsion with 4 drops of the
donor's serum.

3. Treat i drop of donor's red-cell emulsion with 4 drops of his serum.

4. Treat i drop of recipient's red-cell emulsion with 4 drops of his serum. Finally
add i c.c. of salt solution to each of the four tubes, shake gently and place in incubator
for two hours.

Tests 3 and 4 should fail to show either agglutination or haemolysins.
Some prefer to keep the tubes over night in ice-box after the preliminary examina-
tion following incubation.

OCCULT BLOOD

When the presence of blood cannot be recognized by macroscopical
or microscopical methods (occult blood) we must resort to spectro-

OCCULT BLOOD TESTS

2I7

scopic or chemical tests. It is in connection with blood in the faces
that these tests for occult blood are chiefly called for. Before making
such tests on faeces it is advisable to have the patient on a meat-free
and green-vegetable-free diet for two or three days. It is chiefly in
carcinoma or ulcerations of the gastro-intestinal tract that such exami-
nations of the faeces are required.

Haemin Crystal Test (Teichmann).— Prepare a solution of o.i gram each of KI,
KBr, and KCL in 100 c.c. of acetic acid. This is a stable solution. Mix some of
the material with a few drops of the solution on a slide, apply a cover-glass and
warm the material until bubbles begin to appear (gentle steaming), then examine for
dark-brown crystals.

Blood in the Urine. — The most rapid method of detection is by using the micro-
spectroscope. An ordinary hand spectroscope will answer however.

Donogany's test is very satisfactory. To 10 c.c. of urine add i c.c. ammonium
sulphide solution and i c.c. of pyridin. The urine will assume a more or less deep
orange color according to its blood content. The spectrum of alkaline methajmo-
globin or haemochromogen will be obtained. See illustrations under urine.

In making the guaiac or other tests it is a good plan to repeatedly filter the blood-
containing urine through the filter. Then touch a spot on the moist filter with the
guaiac or benzidin solution and then finally drop on this so treated spot a drop or
two of hydrogen peroxide solution.

Blood in Faeces or Gastric Contents.— Take 5 grams of faeces and rub it up
thoroughly in a mortar with 15 c.c. of a mixture of equal parts of alcohol, glacial
acetic acid and ether. Filter through an unmoistened pleated filter-paper re-
peatedly until only 3 to 4 c.c. remain of the filtrate. The fasces filtrate can be
first tested chemically by depositing a few drops in the center of three or four
circles of white filter-paper placed in a Petri dish or upon an ordinary white plate.

Weber Guaiac Test. — The moistened spot is then treated with a few drops of a
freshly prepared alcoholic solution of guaiac resin (about 3^ gram of guaiac resin is
broken up into small fragments and shaken up in about 3 c.c. of alcohol) and finally
there is dropped upon the spot a few drops of a solution of hydrogen peroxide.
Waves of blue color extending out into the moistened filter-paper show a positive
test for blood.

Benzidin Test. — For the benzidin test pour on this faeces filtrate-moistened filter-
paper a few drops of the following solution: 2 c.c. of a saturated alcoholic solution of
benzidin 2 c.c. of solution of peroxide of hydrogen and 2 drops of glacial acetic acid.
(Blue.)

If the aloin test is preferred we treat the filtrate-moistened filter-paper with a
few drops of a 3% solution of aloin in 70% alcohol and then treating the spot with
hydrogen peroxide solution. Brick-red color.

Phenolphthalin Test. — The phenolphthalin test is an extremely delicate one and
will show the pink color at times with certain specimens of water, hence one should
always make a control using the reagents without addition of the suspected blood
material.

In my opinion it has great value as a negative test.

Take 2 c.c. of the ether alcohol acetic-acid filtrate and dilute with 7 or 8 c.c. water.

2l8 MICROMETRY AND BLOOD PREPARATIONS

Neutralize the acidity with sodium hydrate. Then add i c.c. of the phenolphthalin
reagent, mix and finally add several drops of i to 10 dilution of peroxide of hydrogen
and note the formation of a decided rose-pink coloration, varying in depth according
to the amount of occult blood.

To prepare the reagent dissolve i or 2 grams phenolphthalein and 25 grams KOH in
100 c.c. distilled water. Add 10 grams powdered zinc and heat gently until solution
is decolorized. Phenolphthalin is a reduction product of phenolphthalein.

More Reliable is the Spectroscopic Test. — For this we take about 3 c.c.
of the concentrated ether, acetic acid, alcohol faecal nitrate and add to it
2 c.c. of pyridin. Then add not more than 2 to 3 drops of ammonium
sulphide solution. (The ammonium sulphide solution should be kept
in an amber-colored, glass-stoppered bottle. The solution should be
freshly prepared every ten days.) Examine the solution, contained hi a
small test-tube, with the spectroscope and the two absorption bands of
methaemoglobin-alkaline (haemochromogen), between D and E, show
a positive blood test. Comparison should be made with fresh blood,
in which the absorption band in the yellow is nearer line D (oxy-
haemoglobin spectrum).

The great trouble about the spectroscopic test is that it will only show the presence
of quite large amounts of blood. // is by no means a delicate test.

ACIDOSIS AND METHODS FOR ITS DETERMINATION

Everyone is familar with that form of respiratory disturbance
associated with diabetic coma, known as Kussmaul's air hunger. Here
we have hyperpnoea, a form of dyspnoea typically without cyanosis,
and the best clinical evidence of acidosis.

Reduced alkalinity of the blood would be a better expression than acidosis because
even a neutral reaction of the blood would be incompatible with maintenance of life.

Acidosis is an important consideration in alimentary tract disturb-
ances of infants and children, as in infantile diarrhoeas or cyclical
vomiting of children. It is not infrequent in the pneumonias of
children. In adults we must keep the possibility of the occurrence of
acidosis in mind in the vomiting and eclampsia of pregnancy, in sal-
icylate poisoning, following chloroform anaesthesia, and in chronic
nephritis as well as in many infectious diseases.

Sellard's alkaline treatment for the acidosis of cholera is a measure of the utmost
value.

Starvation, whether the result of gastric ulcer, gastric carcinoma or otherwise, is a
recognized cause of acidosis. The insistence upon one-sided diets in children is often

ACIDOSIS 219

the cause of increased acid content of the blood and such causes are not absent in
many of the dietary treatments of diseases of adults, whether in the height of the
disease or during convalescence.

From the laboratory standpoint acidosis is usually recognized by an increase in the
urinary acetone bodies or by noting an increase in the ammonia quotient due to
demands upon the alkali for neutralization of the increased acid production. Ace-
tone, diacetic acid and /3-oxybutyric acid (acetone bodies) come from abnormal met-
abolism of fats. When not formed in excessive amounts the two acids are con-
verted into acetone and appear in the urine as such. With more marked production
acetone formation falls behind and diacetic acid appears or, with still more marked
acid production, /3-oxybutyric acid.

These acids, of themselves, seem harmless and their injurious
action is connected with abstraction of alkali from the blood.

There are some who think lactic acid may be formed from abnormal metabolism
of carbohydrates and that certain cases of acidosis failing to show increase of acetone
bodies may be connected with lactic acid increase. Of course, retention of acids of
normal metabolism would also cause acidosis. To prevent this the organism
utilizes a sufficient amount of the ammonia from protein or rather amino-acid
metabolism instead of further changing it into urea. So that an increased am-
monia quotient may signify that nature has control of the abnormal acid production.

In the acidosis connected with chronic nephritis the preformed
ammonia may be lower than normal indicating some defect in the
mechanism of ammonia neutralization of acids.

Acetone bodies can come from proteins as well as fats. Whatever may be the
explanation of abnormal formation of acetone bodies it seems to be associated with
inability of the organism to obtain sugar for its tissues, hence the therapeutic value
of giving glucose in acidosis conditions where the trouble is carbohydrate deficiency.
Glucose administration is frequently combined with alkali treatment in acidosis.
Of course, where the trouble is an inability on the part of the cells to utilize the sugar,
which may be circulating in greatly increased amounts in the blood, from lack of
pancreatic internal secretion (as in diabetes), sugar injection would have no effect on
this abnormal acid production.

Even in ordinary metabolism great amounts of acid are produced
but these are eliminated normally by way of lungs as well as urine.
In this connection a failure on the part of the kidneys to remove
acid would result in acid retention and, if sufficiently marked, acidosis.

Besides the phosphoric and sulphuric acid produced in the metabolism of the
phosphorus and sulphur of proteids we have enormous amounts of carbonic acid
formed in the tissues.

The alkalinity of the blood is maintained by the bicarbonate of soda,
by sodium and potassium phosphates and to some extent proteins can

220 MICROMETRY AND BLOOD PREPARATIONS

.

neutralize acids. The carbonic acid is taken up by the bicarbonate
of the blood and gotten rid of as CO2 by the lungs, without any loss of
sodium bicarbonate.

Other metabolic acids, however, cause a loss of sodium bicarbonate (and along
with this the other blood alkalis) so that the determination of the lowering of this
salt in the blood indicates an acidosis. This may be carried out by van Slyke's
method described below.

It has been found that the alveolar CO2 falls with a fall in the plasma
bicarbonate. (This however does not hold with cardio-respiratory
cases.) Therefore by determining the CO2 content of the expired air
upon forced expiration we can judge as to reduction of blood carbonate
and of course acidosis.

Sellards' method for determination of serum acidosis is quite simple and reliable.
To carry out the test we add i c.c. of serum, drop by drop, to 25 c.c. of absolute
alcohol (it is very important to secure a neutral alcohol and I have found that of
Merck satisfactory). This precipitates the protein which is the factor interfering
with a sharp end reaction. After filtering we add 3 or 4 drops of neutralized phe-
nolphthalein solution to the fitrate and evaporate the alcohol in a porcelain dish on
a water-bath. Every piece of apparatus must be perfectly dry and the steam vapor
of the bath quite low to avoid the taking up by the alcohol of water. In normal
cases the dark pinkish tinge of the sediment, after evaporation, will remain at least
one hour, while with cases showing increased acidosis the reddish tinging of the
sediment disappears in a few minutes. An electric bath is desirable.

A very simple way of determining bicarbonate diminution is the
test for tolerance of alkalis. The giving of 5 grams of bicarbonate of
soda to a normal person on a mixed diet will bring about an alkaline
urine. Boiling the urine will bring out the alkalinity to litmus more
sharply.

These amounts are increased until the urine becomes alkaline. In some cases of
acidosis massive doses of bicarbonate, as 150 grams in one or two days fail to produce
an alkaline urine.

Plasma Bicarbonate. — Van Slyke has devised an apparatus for the determination
of the carbon dioxide capacity of oxalate plasma.

He uses i c.c. of the plasma which is shaken with air containing 6% of CO2 and
is introduced into the following apparatus. A 50 c.c. pipette-shaped apparatus is
provided at top and bottom with three-way stopcocks and connected with a bulb of
mercury. The pipette is first filled with mercury and the plasma having been intro-
duced followed by i c.c. of water and 0.5 c.c. of N/i acid the mercury is withdrawn
from the pipette by lowering the bulb reservoir, thus creating a Torricellian vacuum.
The CO2 escapes from the solution as the result of a few minutes shaking and the
watery solution is drawn off from the lower cock. The mercury is again made to
fill the pipette by the other entrance of the three-way cock at the bottom and rises

ACIDOSIS TESTS 221

to the level of the CO2. This volume is read off in the calibrated upper stem of
the pipette (calibrated in 0.02 c.c. divisions) and the calculations made accordingly.
Normal serum binds about 75% of its volume of CO2 while in acidosis figures as
low as 20% may be obtained.

For the determination of the CO2 content of alveolar air the apparatus
of Fridericia is quite practical. In this the volume of CO? in 100 c.c.
of alveolar air is obtained. In such methods temperature plays so
important a part in estimating volume that it is hardly applicable
except in the hands of one accustomed to gas analysis.

We have constructed an apparatus, using a Sedgwick aerobioscope as an air
chamber and fitting the ends with glass stopcocks. The patient having expired,
to get rid of air in upper air passages, then with forced expiration fills the 200 c.c.
chamber of the aerobioscope, immediately afterward closing the stopcocks. We
then introduce 5 c.c. of N/i KOH, shaking at intervals for one-half hour to allow
absorption of CO2 by the KOH. We then add a few drops of phenolphthalein as
indicator and titrate in the aerobioscope chamber the loss in alkalinity of the
KOH, using N/i HC1. (For hydrogen-ion concentration method see Appendix.)

Besides the tests for serum acidosis of Sellards and that for carbon
dioxide content of alveolar air we should also determine ammonia
nitrogen output as well as its quotient.

Urinary tests for acidity and acetone bodies content of the urine should also be
made as well as that for alkali tolerance. These tests are all very simple and can be
easily carried out by any well-trained laboratory worker.

[AFTER XIV
NORMAL AND PATHOLOGICAL BLOOD

IN considering what may be termed normal blood, it must be borne
in mind that the normal varies for men, women, and children:

Hb. Red cells Leukocytes

Men, 90 to 110%, 5 to 5^ million, 7500

Women, 80 to 100%, 4}^ to 5 million, 7500
Children, 70 to 80%, 4^ to 5 million, 9000

COLOR INDEX

This is obtained by dividing the percentage of the haemoglobin by
the percentage of red cells, 5,000,000 red cells being considered as
100%.

To obtain the percentage of red cells it is only necessary to multiply the two ex-
treme figures to the left by two. Thus if a count showed the presence of 1,700,000
red cells, the percentage would be 34(17 X 2 = 34). If the Hb. percentage in this
case were 50; then the color index would be 50 -5- 34, or 1.4.

In normal blood the color index is, approximately, i.

In anaemias we have three types of color index: i. The pernicious anaemia type,
which is above i. Here we have a greater reduction in red cells than we have of the
haemoglobin content of each cell. 2. The normal type, when both red cells and
haemoglobin are proportionally decreased, as in anaemia following haemorrhage.
3. The chlorotic type. Here there is a great decrease in haemoglobin percentage,
but only a moderate decrease in the number of red cells. Hence the color index
is only a fraction of i. For example, in a case of chlorosis we have 40% of haemo-
globin and 90% of red cells, 40 -f- 90 = 0.4.

RED CELLS

In considering the corpuscular richness of a specimen of blood, it
must be remembered that this does not necessarily bear any relation to
the quantity of blood in the body. Thus, a more or less bloodless-
looking individual, the total quantity of whose blood is greatly reduced,

222

PATHOLOGICAL RED CELLS 223

may, notwithstanding, give a normal red count. In examining a speci-
men of peripheral blood we get a qualitative, not a quantitative result.

Normally, we have an increase in red cells in those living at high altitudes. An
altitude of 2000 feet may increase the red count about 1,000,000, and a height
of 6000 feet about 2,000,000. Profuse sweats and diarrhoeas also increase the
red count. Pathologically, in chronic polycythemia with cyanosis and splenic
enlargement, we have a red count of about 10,000,000. In cyanosis from heart
disease, etc., and in Addison's disease there is also an increase in red cells.

The normal red cell or erythrocyte measures about 7.5/1 in diameter. It is non-
nucleated and normally stains with acid dyes, taking the pink of eosin or the orange
of orange G. If larger, 10 to 20/1, it is called a macrocyte; if smaller, 3 to 6/i, a
microcyte.

Anisocytosis is a term applied to a condition where marked varia-
tion in size of the red cells occurs.

Macrocytes are rather indicative of severe forms of anaemia, the microcytes, of
less grave types. When the red cell is distorted in shape, it is called a poikilocyte.
Care must be exercised that distorted shapes are not due to faulty technic. Crena-
tion and vacuolation of red cells are marked in poorly prepared specimens.

In addition to variation in size and shape, we also have pathological variation in
staining affinities.

Achromia. — This is characterized by pallor of the central portion of
the stained red cell. It also shows as a central vacuolation in fresh
blood and is apt to deceive one in the anaemic blood of malaria.

Polychromatophilia.— This shows itself by red cells taking a brownish
to a dirty blue tint, as is frequently seen in immature red cells, especially
nucleated ones.

Granular basophilic degeneration (also termed punctate baso-
philia and stippling) refers to the presence of blue dots in the pink back-
ground of stained red cells. It is found in many severe anaemias, as
pernicious anaemia, the leukaemias, malarial cachexia, etc. It is very
characteristic of lead poisoning.

The nucleated red cell, while normal for the marrow, is always
pathological for the blood of the peripheral circulation.

Normoblasts have the diameter of a normal red cell. The nucleus is round and
stains intensely with basic dyes, often appearing almost black. Another character-
istic is that it frequently appears as does the setting in a ring. Some give the term
microblast to smaller nucleated forms. In normoblasts the red cell proper stains
normally. The Wgaloblasts not only have a greater diameter than the normoblast,
but the nucleuses poor in chromatin, stains less intensely and is less distinctly out-
lined. Instead of being round, the nucleus is irregular and may be trefoil in shape.
The cytoplasm surrounding the nucleus shows polychromatophilia. This con-
trasted with the pure blue of the lymphocytes should differentiate.

224 NORMAL AND PATHOLOGICAL BLOOD

Normoblasts are found in secondary anaemias, and especially in
myelogenous leukaemia. Megaloblasts are peculiarly characteristic of
pernicious anaemia. Enormous megaloblasts are sometimes termed
gigantoblasts.

In aplastic anaemia ( a severe type of pernicious anaemia), in contrast to ordinary
pernicious anaemia, nucleated reds are very rarely found. There is also very little
poikilocytosis, and the color index is about normal. It is a rare, rapidly fatal
anaemia, particularly of young women.

It does not show remissions, runs a rapid course, and is attended with a marked
increase of lymphocytes. The bone marrow of the femur is pinkish yellow and
homogeneous.

The term leukancemia has been employed to describe conditions
which partake of the characteristics of pernicious anaemia and leukaemia.

WHITE CELLS

Owing to the conflicting views as to origin, nature, and functions of
the various leukocytes, their classification is in a state of confusion.

As regards the appearance of the cells, this of course varies as the stain used, and
it requires considerable experience for a single individual to be able to positively
recognize the difference between a lymphocyte and a large mononuclear when one
specimen is stained with a Romanowsky stain, another with Ehrlich's triacid, and a
third with a haematoxylin and eosin. This, of course, is intensified when different
persons adhere to the method of staining which they prefer and are at a loss to
appreciate differences which are brought out by some other stain used by some other
person. Even with the same stain used with different specimens of blood we find the
staining characteristics of various leukocytes imperceptibly merging the one into
the other, so that at times it is impossible for one, even with his own standard of
differentiation, to be sure whether he is dealing with a lymphocyte or a large mononu-
clear. The difficulty is even greater when we deal with Tiirck's irritation forms and
with myelocytes.

Without going into the various granule stainings so thoroughly brought out by
Ehrlich, we shall immediately take up the question of a practical classification for use
in making a differential count. As the Romanowsky method of staining (Wright,
Leishman, or Giemsa) gives us information not yielded by either haematoxylin and
eosin or the triacid, the points of differentiation to be referred to in that which
follows is with blood so stained.

In considering the staining affinities of different parts of the leuko-
cytes, it is convenient to divide such into basic ones, acid ones, and
those which may be said to be on the border line between these — the
so-called neutrophilic affinities.

With Wright's stain we have the eosinophile or oxyphile affinity of the
granules of eosinophiles for acid dyes, in this case eosin. The nuclei and baso-

NORMAL LEUCOCYTES 225

phile granules have affinities in greater or less degree for basic stains (the blue and
the violet shading resulting from methylene blue as modified by polychroming).
With the granules in the cytoplasm of the polymorphonuclears and neutrophilic
myelocytes, and to a less extent in the transitional, we have a staining which merges
into a yellowish red on the one extreme and into a lilac on the other. As a standard,
neutrophilic granules should be a mean of these extremes.

Not only by reason of the authority of Ehrlich, but because such a
division gives all variations, which can then be combined by one pre-
ferring a simpler classification, it would seem proper to divide the
normal leukocytes into hyaline and granular cells. Of the former we
have the lymphocytes, the large mononuclears and the transitionals. Of
the latter the polymorphonuclears, the eosinophiles and the mast cells.

HYALINE LEUKOCYTES

1. Lymphocytes. — As a rule, the cells of this type are about the
diameter of a red cell. The nucleus is generally quite round but may
show one or more bulging processes. The large lymphocytes are rare
in the blood of adults but make up about 10% of the leukocytes of young
children. In making a count it is best to group large and small lympho-
cytes under one heading but for distinction we may divide them into:

(a) Small Lymphocytes. — These are small round cells about the size
of a red corpuscle with a large centrally placed, deeply violet staining
nucleus and a narrow zone of cytoplasm. This cytoplasm may not be
more than a mere crescentic fringe. This is the type of lymphocyte
which makes up the greater proportion of the leukocytes in chronic
lymphatic leukaemia. At times these cells seem to be composed of
nucleus alone.

(b) Large Lymphocytes. — These are of the same type as small lympho-
cytes, but possessing more cytoplasm. The nucleus, while round and
taking a fairly deep rich violet stain, does not stain so deeply as the
nucleus of the small lymphocytes. The cytoplasm is a clear, trans-
lucent, pure blue. It may contain pinkish granules known as azur
granules, but these are of rather large size and do not mar the glass-
like appearance. They are from 9 to i$n in diameter and are com-
mon in children. In the acute lymphatic leukaemias they at times
predominate.

2. Large Mononuclears.— These are large round or oval cells with
a nucleus which has lost the richness of violet staining of the lymphocyte
nucleus. The nucleus is furthermore frequently irregular in outline
or may show the commencing indentation of the transitional nucleus.

is

226 NORMAL AND PATHOLOGICAL BLOOD

There is not that sharp distinction between nucleus and cytoplasm that exists in
the lymphocytes. The cytoplasm of the large mononuclear gives the impression of
opacity, as if it were frosted glass instead of clear glass. The neutrophile mottling
which begins to appear causes a disappearance of the pure blue character of the cyto-
plasm of the lymphocyte. It is principally by the washed-out staining of the nucleus
and the opaque lilac of the cytoplasm that we differentiate them from the lympho-
cytes. They greatly resemble Turck's irritation forms or plasma cells and may be
confused with myelocytes.

3. Transitionals. — These appear as but a later stage in the decay
of the large mononuclears; the nucleus is more indented, frequently
horseshoe-shaped, and has a washed-out violet shade of less intensity
than that of the large mononuclears. These are the cells so often dis-
rupted in smears. The old view that the transitional was the pre-
cursor of the polymorphonuclear has few advocates at the present
time.

While it may be convenient to consider hyaline cells as representing different stages
in development, yet from a standpoint of immunity this is untenable. The large
mononuclears and transitionals are the cells in which we find certain animal cells
and pigment phagocytized, as is the case in malaria. These cells are the macro -
phages of Metchnikoff and are probably derived from the bone marrow.

In the tropics one of the most important points in a differential
count is the matter of an increase in the large mononuclears and
transitionals, both of which seem to respond to the same stimulus,
which is most commonly malaria but may also be from other protozoal
infections.

From a practical standpoint I always group them together and as a matter of fact
it is difficult to separate a large mononuclear showing considerable irregularity of
nucleus from a transitional with less marked nuclear indentation.

The lymphocytes take origin from the lymphoid tissue, and very
probably the large lymphocyte is a younger, more immature cell than
the small lymphocyte.

Ehrlich and Naegeli regard the large mononuclears as of myeloid origin while
Pappenheim considers them to belong to the group of lymphocytes.

A normal percentage of large mononuclears and transitionals com-
bined should not exceed about 4%.

GRANULE CONTAINING LEUKOCYTES

In addition to the series of leukocytes just considered we have present
normally in the blood three types of granular cells distinguished
according to the staining affinity of their granules. These are:

ARNETH INDEX 227

1. Polymorphonuclear Leukocytes.— This cell normally constitutes
the greater proportion of the leukocytes. It is an amoeboid, actively
phagocytic cell, about 10 or i2/* in diameter, and is the microphage of
Metchnikoff.

Bacteria are actively phagocytized by this cell, and it is the cell concerned in
determining the opsonic power of blood to various bacteria. It has fine lilac gran-
ules which are termed neutrophilic (epsilon granules). The single nucleus is rich in
chromatin and is lobose like the kernel of an English walnut; frequently it resembles
the letter z. These cells are derived from the neutrophilic myelocytes of the bone
marrow. It is in these cells that the glycogen, or iodophil granules, appear in certain
suppurative conditions.

Arneth Index.— A great deal of interest has been aroused in the so-called Arneth
index, especially in connection with prognosis in tuberculosis and various pyogenic
infections. The basis of the test is that polymorphonuclears showing only one or
two nuclear nodes are considered immature while those having three, four or five
nuclear nodes possess greater phagocytic power.

A normal distribution is as follows:

Class 1 Class II Class III Class IV Class V

6% 35% 42% 16% i%

To obtain the Arneth index add to the sum of the polymorphonuclear percent-
ages of cells containing one and two nodes one-half of the percentage of those having
three nodes. In the above we have as the normal Arneth index 62.

In an advanced case of tuberculosis we might have an index of 79, obtained as
follows :

Class I Class II Class III Class IV Class V

20% 45% 28% 6% i%

2. Eosinophile Leukocytes. — These are very striking cells with
coarse granules staining brilliantly pink, the eosinophile, oxyphile, or
acidophile granules (alpha granules of Ehrlich). The cells are a little
larger than the polymorphonuclears.

The normal eosinophile is to be distinguished from the eosinophilic myelocyte by
possessing two distinct lobes in the nucleus. At times we find three nuclei. The
nucleus of the myelocyte is round. The eosinophile is the cell so frequently increased
in infections by intestinal animal parasites.

3. Mast Cells. — These also have coarse granules, but they stain a
deep violet blue. Hence they are basophile granules (gamma granules) .
In fresh blood these granules do not show up very well, thus they can be
distinguished from the highly refractile granules of the eosinophile.

228 NORMAL AND PATHOLOGICAL BLOOD

The trilobed nucleus stains less intensely than the granules. As a rule,
the mast cell is about the size of a polymorphonuclear.
In a differential count of normal blood we find about the following percentages.

Polymorphonuclears, 65 to 70%, about 5000 per cu. mm.
Small lymphocytes, 20 to 30% about 1500 per cu. mm.
Large lymphocytes, 2 to 6%, about 200 per cu. mm.
Large mononuclears, i to 2%, about 100 per cu. mm.
Transitionals, 2 to 4%, about 200 per cu. mm.

Eosinophiles, i to 2%, about 100 per cu. mm.

Mast cells, M to >£%, about 25 per cu. mm.

NOTE: The lymphocyte percentage of infants is about 60.

DIFFERENTIAL COUNT
«
In making a differential count I would recommend the following

from the directions of Schilling-Torgau.

It will be remembered that considerable interest was raised a few years ago in what
was termed the Arneth index. In this the more normal, more mature, better resist-
ing polymorphonuclears were considered to have three or four lobes to the nuclear
structure, even occasionally five. The immature cells had only one or at most two
lobes to the nucleus. The index was obtained by adding the percentages of cells
showing one and two lobes to one-half the percentage of those with three lobes. As
will be understood a high percentage of these immature cells was unfavorable in prog-
nosis. These cells are graded from left to right I, II, III, IV, V, as to separate
masses in the nucleus, so that when the percentage is shoved or displaced to the left
it indicates an increase in the immature cells.

Schilling-Torgau divides his polymorphonuclears into i. the myelocyte which is
always of course a pathological cell; 2. the immature form polymorphonuclear. In
this there is a close resemblance to the neutrophile myelocyte but there is a nuclear
indentation instead of the round nucleus of the myelocyte. It is this cell which often
puzzles us as to whether to regard it as a true myelocyte. It is the metamyelocyte
of many authorities. 3. Between the mature or segmented polymorphonuclear
and the immature one or metamyelocyte we have what may be designated the band
form nucleated one. These show the type of nucleus which one is familiar with in
the nucleus of the transitional. 4. The mature, multilobed or segmented nucleus of
the typical polymorphonuclear.

It would seem that if all laboratory workers would agree upon some
single method of recording differential counts it would be advantageous.

In the differential count he not only divides up the polymorphonuclears but
makes no separation of small from large lymphocytes. Although I have always
divided lymphocytes into large and small ones I believe it unnecessary and imprac-
tical and shall henceforth group all such cells in one grouping. The statement that
large mononuclears and transitionals are cells of a similar origin, type and significance
has always been my idea.

PATHOLOGICAL LEUCOCYTES
SCHEME OF SCHILLING-TORGAU

229

Type of cell Normal Percentage ,

percentage

1. Mast cells. x x 0

2. Eosinophiles. , i e

f a. myelocytes. o 0.5

Neutro- ' b' immature fonns (metamye-

' ~

philes ocyes. o 5.0

I c. bandform (Stabkernige). 4 I3.5

[ d. multilobed (Segmentkernige) . 63 64.0

4. Lymphocytes. 23 10.5

5. Large mononuclears and transitionals. 6 4.0

PATHOLOGICAL LEUKOCYTES

The leukocytes which are found in the peripheral circulation only
in pathological conditions are:

1. Neutrophilic Myelocytes. — The common type is a large cell
with a large centrally placed, feebly staining nucleus.

This may be recognized by the difficulty of distinguishing the nucleus from
the cytoplasm, there being no sharp line separating these parts of the cell. They
imperceptibly merge into one another. They differ from a large mononuclear in
that the cytoplasm is distinctly dotted with neutrophile granules and that we
cannot make out a distinct line of separation of a slightly irregular or indented
nucleus from the surrounding slightly neutrophilic cytoplasm. Cornil has described
a very large myelocyte with eccentrically placed nucleus and neutrophilic granules.

Myelocytes are at times found with both basophilic and neutrophilic
granules, and may rarely be seen to have all three kinds of granules on a
single myelocyte, acidophile, basophile, and neutrophile.

2. Eosinophilic Myelocyles. — These can be distinguished from
normal eosinophiles by their possessing a single round nucleus, not
bilobed. These myelocytes may be as large as a normal eosinophile,
but frequently are no larger than a red cell.

The neutrophile myelocyte is characteristic of spleno-myelogenous leukaemia, the
eosinophile one of myelogenic leukaemia. The occurrence of an occasional myelocyte
is frequently noted in conditions having a leukocytosis. In diphtheria their presence
in numbers is of bad prognostic import. Myelocytes are of diagnostic importance
in metastases of malignant tumors.

3. The Irritation Cell of Tiirck, or Plasma Cell. — This cell has a
faintly staining, eccentrically placed nucleus, and a dark opaque blue,
frequently vacuolated, cytoplasm. They are usually recorded as large

230 NORMAL AND PATHOLOGICAL BLOOD

mononuclears. Tiirck supposed them to appear in the circulation as
the result of bone-marrow irritation.

4. Myeloblasts. — These cells are found in myeloid leukaemia and
though often mistaken for lymphocytes or large mononuclears they are
of marrow origin. They are the lymphoid cells of the marrow and are
the parent cells of myelocytes. The nucleus stains more intensely
than that of the large mononuclear and the cytoplasm is more deeply
blue stained than that of the large lymphocyte. They also contain
three or four nucleoU.

Pyronin methyl-green staining is best for demonstrating the nuclei.

5. Pathological Large Lymphocytes. — These are, as a rule, much
larger than normal large lymphocytes and show poorer staining of both
nucleus and cytoplasm. The nuclei often show the appearance of
division into two or more lobes, thus showing the characteristics of
Rieder cells.

They may be confused with large mononuclears but are considered to be derived
from the germinal centers of various lymphoid tissues. They are found in leukaemic
and pseudo-leuksemic conditions.

6. Megakaryocytes. — These are the giant cells of the bone marrow
and are but rarely found in the blood. The nucleus is gnarled.

BLOOD PLATELETS

These are normally present in blood in the number of about 350,000 per cubic milli-
meter. They disintegrate very quickly after the blood is withdrawn. Wright has
demonstrated that they are pinched-off projections of giant cells of the bone marrow.
They consist only of protoplasm, no nuclear material. They do not contain haemo-
globin. In conditions where giant cells are less abundant, as in pernicious anaemia,
the blood platelets are less abundant. In myelogenous leukaemia they are very abun-
dant. They vary in size from 2 to 5/1 according as a larger or smaller pseudopod of a
giant cell has been broken off. Stained with Wright's stain, they are more purplish
than blue and show thread-like projections. They are often mistaken for the proto-
zoal causes of various diseases. Especially are they confused with malarial para-
sites when lying on a red cell. The blood plate has no brick-red chromatic material;
it is purplish rather than blue, and has no pigment grains. It is advisable to com-
pare these isolated blood plates with the larger or smaller aggregations scattered
about the smears. In this way their true character is apparent. In addition to
blood platelets, which in fresh blood can only be observed when a fixative is used, we
have other confusing bodies.

The hamokonia of Muller are small, highly refractile bodies showing active
oscillatory movement. They are supposed to be cast-off granules of eosinophiles or

EOSINOPHILIA 231

other leukocytes, or possibly derived from nuclei. As this blood dust or haemokonia
is found in a marked degree in lipaemia it may be that the particles are fat. It is
interesting that this lipaemia is absent after the taking of large quantities of fat in
cases with serious pancreatic trouble. The serum of a normal individual is rather
turbid after slight indulgence in butter. Pinched-off fragments of red cells may
also appear as possible protozoal bodies.

LEUKOPENIA

This is a term used to designate a reduction in the normal number
of leukocytes. A leukocyte count of 5000 would represent a slight
leukopenia; one of 2000, a marked leukopenia. In the latter stages
of typhoid, and in acute miliary tuberculosis, we expect a moderate
leukopenia. Glandular tuberculosis may give a very marked leuko-
penia. Tuberculous peritonitis will show moderate leukopenia or a
normal count.

The leukopenia of typhoid is moderate and is often preceded in the first few days
by a moderate neutrophile leukocytosis. Later on we have a decided increase in the
lymphocytes. A marked diminution or absence of eosinophiles is so characteristic
that any increase in eosinophilic percentage negatives a diagnosis of typhoid.

Paratyphoid gives a similar blood picture.

Chronic alcoholism and chronic arsenic poisoning cause a reduction in the number
of the white cells. Pernicious anaemia especially the aplastic type shows a marked
leukopenia, as is also the case with Banti's disease. Two tropical diseases, kala-azar
and dengue, show a marked leukopenia, the counts often being below 2500. During
the apyrexial period of malaria we may have a white count of 5000.

It has recently been claimed that a leukopenia with a coincident
marked reduction in the lymphocytes is characteristic of measles and
that this occurs several days before the Koplik spots appear.

Kocher notes that in exophthalmic goiter the leukocyte count is considerably
diminished and that the polymorphonuclears are not much more than one-half the
usual percentage while the percentage of the lymphocytes is almost double the
normal.

X-ray treatment tends to destroy leukocytes in the exposed region, especially
polymorphonuclears. The small lymphocytes are least affected.

EOSINOPHILIA

Where the eosinophiles are increased to 5%, we have a moderate
eosinophilia. In some cases of infection with intestinal parasites,
especially hook-worms, but also from other parasites, as round and
whip-worms, we may have an eosinophilia of 30 to 50%. In Guam,
among the natives, it is difficult to find an eosinophile count under 15.%

232 NORMAL AND PATHOLOGICAL BLOOD

The eosinophilia tends to disappear when the anaemia becomes very
severe.

Echinococcus infection has an eosinophilia which disappears when the cyst is
removed. Continuance of the eosinophilia indicates that all cysts were not gotten
rid of.

The eosinophilia of trichinosis is best known, and a combination of
this blood finding with fever and marked pains of muscles, would
justify the excision of a piece of muscle for examination for encysted
embryos.

In true asthma eosinophilia is marked, and its absence is of value in indicating
other causes for the condition. Certain skin diseases, especially pemphigus, show
eosinophilia. Blastomycoses are usually found to show eosinophile increase.

Eczema and psoriasis are not apt to give more than 3 or 4% eosino-
philes. A rather high degree of eosinophilia is found in mycosis
fungoides.

Scabies also gives an eosinophilia.

The proportion of eosinophiles in the blood of children is greater
than in that of adults.

Increase of both eosinophiles and mast cells is found in myelogenous leukaemia.

An eosinophilia tends to appear following splenectomy. With a Wright stain
showing acid tendencies one may count polymorphonuclears as eosinophiles unless
noting smaller size of granules.

LEUKOCYTOSIS

It is to an increase in the polymorphonuclears that this term is
usually applied, the term lymphocytosis or eosinophilia being employed
where white cells of eosinophile or lymphocyte nature are increased.
We have physiological leukocytosis in the latter weeks of pregnancy,
also in the new-born, and in connection with digestion.

Pathological Leukocytosis.— Pneumonia. In this disease we have a
leukocytosis of 20,000 to 30,000 or higher. The eosinophiles are al-
most absent. A normal leukocyte count in pneumonia makes a prog-
nosis unfavorable.

The leukocyte count drops about the time of the crisis, and with the reappearance
of eosinophiles is a favorable sign.

Toxaemic conditions as uraemia, diabetic coma and poisoning by CO2 tend to show
a leukocytosis.

LEUCOCYTOSIS

233

Septic processes. The leukocyte count is of great value, especially
when we obtain a leukocytosis with 80 to 90% of polymorphonuclears,
as in appendicitis, cholecystitis, or other suppurative conditions.
Read article under the blood cultures, Chap. XXIX. A marked
leukocytosis is of diagnostic importance in acute ulcerative endocarditis
provided it is not fulminant in type.

FIG. 55.— Leukocytosis (40,000); sixteen polymorphonuclears in field. (Cabot.)

According to Cabot, leukocytosis varies in infections as follows:

1. Severe infection— good resistance; early, marked and persistent leukocytosis.

2. Slight infection — slight resistance; leukocytosis present, but not marked.

3. In fulminating infections we may have no increase in whites, but a higher per-
centage of polymorphonuclears.

4. Slight infection and good resistance may not be productive of leukocytosis.

It is in connection with the question of operation in appendicitis or similar condi-
tions that the matter of a leukocyte count is of prime importance. If there be a
leukocytosis but with less than 75% of polymorphonuclears it indicates an infection
of little virulence or a walled-off process with an exacerbation. It is difficult to form
an opinion when the polymorphonuclears are under 80%. Leukocytosis with poly-
morphonuclear percentage of 85 to 90 indicates immediate operation; percentages
over 90 point to peritonitis and if with such percentages of polymorphonuclears there
is absence of leukocytosis the prognosis is grave.

The blood of cases with malignant tumors tends to show a moderate
leukocytosis except in epithelioma of the skin. When a cancer is
ulcerating quite a high white count may be obtained.

Spirochaete fevers, as relapsing fever, may give a leukocytosis of from 25,000 to
50,000.

Smallpox, especially at time of pustulation, plague, scarlet fever,
and liver abscess give a leukocytosis of from 12,000 to 15,000.

234 NORMAL AND PATHOLOGICAL BLOOD

Smallpox often shows a very large percentage of very characteristic large
nuclears.

The leukopenia and lymphocyte increase in measles are important points in
differentiating it from scarlatina.

Influenza shows a leukopenia at first, then a leukocytosis and, follow-
ing the fall in fever, a second lowering.

With meningitis counts of 25,000 are not unusual, in abscess of the brain the white
count rarely exceeds i5,'ooo.

Poliomyelitis and polioencephalitis give a slight leukocytosis during
the febrile accession.

Erysipelas and epidemic cerebrospinal meningitis also give a leukocytosis of from
1 5,000 to 20,000. In malignant diseases we sometimes have a moderate leukocytosis.
Rogers states that in liver abscess, with a leukocytosis of 15,000 to 20,000, we have
only about 75 to 77% of polymorphonuclears — there being also a moderate increase
in the percentage of large mononuclears.

Drugs such as antipyrin may give a leukocytosis. The leukocyte increase of
pilocarpine is rather a lymphocytosis. Cinnamate of soda, sodium nucleate bacterin
injections and turpentine have been used in kala-azar to increase leukocytes.

LYMPHOCYTOSIS

Of course, the disease in which we have the most marked lymphocy-
tosis is lymphatic leukaemia.

The lymphocytosis of typhoid fever has been taken up under leukopenia.
Whooping-cough may give a lymphocytosis of 20,000 to 30,000.

Young children have normally an excessive proportion of lymphocytes even to a
reversal of the polymorphonuclear-lymphocyte relation of adults. This is apt to be
particularly marked in hereditary syphilis. Enlarged tonsils may give rise to a
lymphocytosis of 10,000 to 15,000 when more than 50% of the white cells will be
lymphocytes. Rickets and scurvy give a lymphocytosis.

In pellagra there is a moderate lymphocytosis, averaging 34% in about a normal
count.

Varicella and mumps may also give an increase in the percentage of
lymphocytes.

Malta fever is a disease which may show quite a lymphocyte increase, this going
with a reduction in polymorphonuclears.

INCREASED LARGE MONONUCLEARS

In tropical work we combine the large mononuclears and transitionals
in a differential count. They are the phagocytes of animal cells or

PRIMARY ANEMIAS 235

parasites. The disease in which their increase is best recognized
is malaria and an increase to 15% where the blood shows moderate
leukopenia is very significant. The melaniferous leukocytes of malaria
are cells of this type.

Other protozoal infections, as kala-azar, trypanosomiasis and amcebiasis cause it.
Filterable virus diseases may show a mononuclear increase, thus yellow fever and
dengue both give an increase about the fifth or sixth day.

In Banti's disease there is an increase in cells of this type and a transitional in-
crease is reported for Hodgkin's disease.

DISEASES IN WHICH THERE is A NORMAL LEUKOCYTE COUNT

Uncomplicated tuberculosis, influenza, Malta fever, measles, try-
panosomiasis, malaria, syphilis, and chlorosis.

In malaria we have a leukocytosis at the time of the rigor, while during the apyrexial
period there is a moderate leukopenia. In malaria we have a marked increase in the
percentage of the large mononuclears and transitionals. These may form from 25%
to 35% of the leukocytes. When bearing particles of pigment they are known as
melaniferous leukocytes — macrophages which have ingested malarial material. In
dengue, at the time of the terminal rash, we may have as great a percentage of large
mononuclears. In this disease, however, we have a great diminution of polymor-
phonuclears from the start (25 to 40%). Instead of a large mononuclear we have
at the onset a lymphocytic increase. There is an increase of large mononuclears in
trypanosomiasis.

The white count is about normal in uncinariasis (Ashford's average
was 7800). Some have reported a. leukopenia in severe cases.

While eosinophilia is the most marked feature in hook-worm disease yet in very

severe cases it may be absent.

•-

THE PRIMARY ANAEMIAS

Chlorosis.— In chlorosis it is the reduction of haemoglobin with the
slight numerical variation from normal of the red cells that makes for
a diagnosis. The color index is very low. There is nothing abnormal
about the leukocytes. Microcytes may be present, and very occasion-
ally a normoblast. Macrocytes and megaloblasts are always absent.
Blood of chlorotics is very pale and very fluid and coagulates rapidly,
hence frequency of thrombosis.

Spleen, liver, and lymph glands as a rule normal.

Simple Primary Anaemia.— This condition is not recognized by many authors,
but is a convenient term under which to group anaemias which are neither chlorosis

236 NORMAL AND PATHOLOGICAL BLOOD

nor pernicious anaemia and for which no assignable cause can be designated. It
is a secondary anaemia without a cause. In it color index is about normal, there is
no change in the leukocytes and cases go on to recovery.

Pernicious Anaemia. — In pernicious anaemia we obtain a very
fluid, but normally colored drop of blood upon puncture. The yellow
marrow of the long bones is transformed into a soft, bright red lymphoid
tissue, smears from which show great numbers of megaloblasts.

Areas of fatty degeneration are characteristic, especially the tiger-lily spots in
the heart muscle. Iron-containing pigment (hemosiderin) is found in the liver,
spleen, and kidneys. Areas of degeneration in the spinal cord may account for nerv-

FIG. 56. — Pernicious anaemia. M.m, Megaloblasts; n, normoblast; s, stippling
(punctate basophilia). (Cabot.)

ous symptoms. The red cells frequently fall below 2,000,000 with patients going
about. Cases have been reported with counts under 200,000. The color index is
high. Megaloblasts are the most characteristic qualitative change in the red cells.

Megaloblastic crises may at certain times show enormous numbers
of megaloblasts. Cases often present remissions in which no megalo-
blasts can be found. In such cases the presence of many macrocytes
should prevent an examiner's reporting against a pernicious anaemia
previously diagnosed.

Poikilocytosis, polychromatophilia, and stippling are also features of the disease.
Normoblasts are far less frequent than megaloblasts and there is usually a moderate
lymphocytosis. Myelocytes may be present, but their precursors, the myeloblasts,
are probably more frequently met with.

Cases of pernicious anaemia show remissions during which the patient is appar-
ently on the road to recovery. Such improvements are only temporary. The
remissions may last from two months to possibly three or four years. Especially

SECONDARY ANAEMIAS 237

in the anaemia of Dibothriocephalus latus do we have a picture of pernicious anaemia.
It is supposed to be due to a toxin present in the heads of these tape-worms.

In pernicious anaemia there is usually an absence of free HC1, which
causes it often to be confused with gastric cancer. Cases of chronic
nephritis are also often much like pernicious anaemia but most difficult
of differentiation are affections of the spinal cord, such as tabes, etc.,
the neurological manifestations of pernicious anaemia causing the
confusion.

Blood changes more or less like those of pernicious anaemia have at times been
noted in children with tuberculosis of bovine nature. The human strain of T.B.
does not seem to produce such changes.

An acute disease showing a rapidly developing anaemia of the pernicious anaemia
type is Oroya fever in which the bone marrow seems especially involved.

SECONDARY ANEMIAS

These are the anaemias which can be definitely traced to some dis-
ease not of the haemopoietic system.

There are two main groups — those following haemorrhage and those
secondary to various diseases. If the haemorrhage is sudden and great,
the resulting condition is one of oligochromaemia — chlorotic in type.
Normoblasts are usually found after the third day.

The low Hb. percentage is apt to continue for several weeks. There is also an
increase in the percentage of polymorphonuclears.

It is a question whether prolonged operation or those requiring narcosis are
justified where the reduction in Hb. is under 40%. (According to Miculicz, 30%
is the minimum).

Where the loss of blood is gradual, as in gastric cancer or severe haemorrhoids
the picture may more nearly approach that of pernicious anaemia. Secondary
anemias usually show a moderate leukocytosis. In chronic nephritis and prolonged
suppurative conditions normoblasts and macrocytes are rare— moderate poikilo-
cytosis with the presence of many microcytes being the rule.

In fatal ansmia from chronic acetanilide poisoning high color index, macrocytes
and megaloblasts have been noted.

In some secondary anaemias, as in syphilis, carcinoma, and tuber-
culosis, we have a chlorotic color index (chloro-anaemias).

In secondary anaemias polychromatophilia, poikilocytosis, and punc-
tate basophilia (stippling) may be present. This latter is very marked
in lead poisoning, but in certain cases of malarial cachexia it may be
equally prominent. The only form of nucleated red cell seen is the
normoblast, in very small numbers, or it may not be present.

238 NORMAL AND PATHOLOGICAL BLOOD

Megaloblasts are practically never seen, except in some of the very severe para-
sitic anaemias, as the broad Russian tape-worm infection. The red cells generally
number between 2,000,000 and 4,000,000, thus differentiating chlorosis. The
leukocytes are frequently increased to 15,000. In the anaemia of splenic anaemia
there is a marked leukopenia. In anaemias from malignant tumors the color index
is usually of the chlorotic type — the haemoglobin content of the red cells being more
affected than the number. Normoblasts are usually present, and this finding may
differentiate gastric cancer from ulcer. In bone marrow metastases megaloblasts
may be expected. Myelocytes and so-called tumor cells (large cells with faintly
staining vacuolated nuclei and but little cytoplasm) may also be found. As a rule,
there is a moderate leukocytosis in malignant disease. Eosinophiles may be largely
increased in sarcoma.

THE LEUKAEMIAS

It is in the leukaemias that we have the greatest increase in the num-
ber of white cells. These cases show more or less anaemia, but we may
have cases of myelogenous leukaemia showing 250,000 leukocytes per
cubic millimeter without particular change in the red cells. The more
marked the red-cell change the more severe the condition.

There are two well-defined types of leukaemia, the lymphatic and the spleno-
myelogenous. It must be borne in mind, however, that while a greater change in
the lymphatic glands may produce the lymphatic type, yet even in such cases we
expect to find alteration in bone marrow and spleen; that is, there is a general involve-
ment of the haemopoietic system in all leukaemias, the activity being most marked in
spleen and bone marrow in certain cases and in lymphatic glands in others.

Myelogenous leukaemia is a very rare disease, about five times as
rare as pernicious anaemia. Lymphoid leukaemia is still more rare.

Splenomyelogenous Leukaemia (myeloid leukaemia). — The differen-
tiation of the blood picture of this disease from leukocytosis does not
depend on the number of leukocytes, but on the presence and large
proportion of myelocytes. We expect both neutrophilic and eosino-
philic myelocytes in myeloid leukaemia — the proportion of these varies,
but, as a rule, the neutrophilic one is the common one. The blood in
advanced cases is milky and shows a most marked buffy coat. The
marrow is largely replaced by a yellow pyoid material. The spleen
may weigh 10 pounds.

The leukocyte count is on the average from 200,000 to 500,000. Cases are re-
ported of more than 1,000,000 white cells. The neutrophilic myelocytes make
up about 30 to 40% of these and, about equal in number, are found the polymor-
phonuclears, while the percentage of the lymphocytes is decreased (2 to 5%) and
normal eosinophiles, eosinophilic myelocytes, and large mononuclears make up the
remaining percentages. We usually have great numbers of normoblasts. Megalo-

.

LEUKAEMIA

239

blasts may rarely be found. The red count is usually about 2,500,000 and the
color index low.

Lymphatic Leukaemia.— In this we have glandular enlargements,
but not such large masses as in Hodgkin's disease. • The red cells are
usually reduced about one-half and the color index is a little below
normal. Normoblasts are rarely found. Myelocytes, as a rule, are
absent, but may amount to 5% of the leukocytes. The predominating
leukocyte (75 to 98%) is the small lymphocyte. In acute lymphatic
leukaemia the large lymphocytes predominate.

FIG. 57. — Myelogenous leukaemia, m, Myelocyte; p, polymorphonuclear; b, mast
cell; n, normoblast. (Cabot.)

These however are pathological and differ from the large lymphocyte in not
having azur granules and the nucleus stains poorly and is often indented. The
leukocyte count is never so great as in myeloid leukaemia, rarely exceeding 125,000.

Pseudoleukaemia.— Hodgkin's disease is usually considered as a
disease with marked glandular enlargements, but with a negative blood
picture, or at any rate only a moderate leukocytosis with a relative
increase of lymphocytes.

The glands in this condition do not soften and on section show diffuse hyper-
plasia of cells of the endothelial type. Eosinophiles are also abundant in the sec-
tions. Connective-tissue increase and small necrotic areas are also features.

The red cells are usually above 3,000,000. It has been considered that an in-
creased percentage of transitionals (10 to 15%), should a leukopenia coexist, is
characteristic.

Undoubtedly the view that so-called lymphosarcomata, lymphatic
leukaemia, and Hodgkin's disease merge into one another and that they

240 NORMAL AND PATHOLOGICAL BLOOD

represent a malignant cell formation in the haemopoietic system is
conservative one to take.

A certain proportion of cases of Hodgkin's disease, however, show endothelial
cell proliferation and a chronic fibroid change.

In Kundrat's lymphosarcoma we have a neutrophile leukocytosis
and a diminution of the lymphocytes. The spleen and liver are rarely
involved.

Another condition with swelling of the lymphatic glands, which do not however
fuse, is the so-called granulomatosis.

FIG. 58. — Lymphatic leukaemia. />, polymorphonuclear; m, megaloblast; e, eosino-
phile. Twenty-one lymphocytes in this field. (Cabot.)

In this we have a polymorphonuclear leukocytosis of from 20,000 to 50,000 with
an increase in the percentage of eosinophiles. The lymphocytes are absolutely and
relatively decreased. In granulomatosis there is no tendency to haemorrhage.

Splenomegaly. — The best known anaemia associated with splenic
enlargement is Band's disease.

Banti's disease also has a very low color index and leukopenia. In
this the primary affection is of the spleen which becomes greatly en-
larged. The accompanying cirrhosis of the liver with its symptoms of
ascites, etc., differentiate it. Splenectomy often cures the disease.
The leukopenia is one showing not only a diminution of polymorphonu-
clear percentage but of cells of the lymphocyte type as well.

There is a considerable increase in the large mononuclear percentage. Nucleated
reds and myelocytes are invariably absent. It must be remembered that we have a
group of cases showing splenomegaly which are syphilitic in origin and which, as a
rule, give a positive Wassermann. Clinically or haematologically they resemble true

SPLENIC ANAEMIA 241

Banti's disease but pathologically the spleen shows a fibrosis instead of the marked
increase in lymphatic tissue characteristic of Banti's disease.

In the tropical splenomegaly or kala-azar we have a marked leukopenia with
a marked reduction in the percentage of polymorphonuclears. The Gaucher type
of splenic anaemia does not show as pronounced and early an anaemia as in Banti's
type.

Certain conditions which partly resemble myelogenous leukaemia
and partly pernicious anaemia are designated leukanaemia. Some con-
sider this to belong to the group of diseases in which the multiple
myeloma is placed.

In splenomegalic polycythaemia we have a red count of from 9,000,000 to 10,000,000.
The Hb. percentage may be 200. There is also a leukocytosis up to 50,000. Pa-
tients are cyanosed and have a very large spleen.

Splenic anaemia of infancy usually occurs between the ages of twelve and twenty-
four months. The spleen is notably enlarged and in many cases the liver is equally
so. The red cells are not greatly diminished in number, 2,500,000 to 3,000,000
being usual findings. Nucleated reds are abundant. While a leukocytosis of
30,000 to 50,000 is often present it is markedly less than that of splenomyelogenous
leukaemia and the increase in white cells is more of those of lymphocyte type.

The color index is very low.

Another splenomegaly of children, clinically resembling kala-azar, is caused by
Leishmania iufantum.

16

PART III
ANIMAL PAR ASITO LOGY

CHAPTER XV

GENERAL CONSIDERATIONS OF CLASSIFICATION AND

METHODS

ANIMALS that are in all respects alike we term a Species. Of course
the male and female of a species may be very unlike, but as a result of
mating they produce young having characteristics similar to the
parents. Now, if, as in the case of the mosquitoes causing yellow fever,
we find some with straight silvery lines and others uniformly showing
crescentic silvery bands about thorax, yet resembling each other closely
in the respect of being dark, brilliantly marked mosquitoes, we should
consider them as being separate species with a certain relationship
to which the term Genus is applied.

The term "genus" is of wider application than the word "species." Thus
animals which agree in the main characteristics of size, proportion of parts, and
general structure are placed in the same genus.

In naming a species we always first write the name of the genus
which has a Greek or Latin name, commencing with a capital, and
follow with the specific name, which latter commences with a small
letter. Thus we designate the dark silver-marked mosquitoes as
belonging to the genus Stegomyia; those showing the characteris-
tics of curved silver bands and two central parallel lines (lyre pat-
tern) on dorsal surface of thorax we designate as Stegomyia calopus;
the species with only the straight silver lines we call Stegomyia
scutellaris.

The specific name may be a noun in the genitive. If an adjective it must agree in
gender with the generic name.

It is permissible to have a masculine noun as a specific name with a feminine
generic name.

243

244 CONSIDERATIONS OF CLASSIFICATION AND METHODS

If the specific name is a modern patronymic we add i in the case of a man or ae for
a woman to the exact and complete name of the person.

Again, certain genera show resemblances which enable us to make broader
groupings to which we apply the term Subfamily. Thus the genus Stegomyia
and the genus Culex have the similar characteristics of palpi in the female being
shorter than the straight proboscis; we therefore classify all species of Stegomyia
and all species of Cidex under the designation Culicinae. The name of a subfamily
ends in "inae." Now, again, certain insects are different from others in having
scales on the wings. We find that not only do the Culicinse have such character-
istics, but the same is observed with the Anophelinae and other similar scale-wing
insects. All of these we term a Family and we speak of the Culicidae, meaning
the family of mosquitoes. The name of a family ends in "idae." Many families
are not subdivided into subfamilies, but are directly separated into genera. Again,
a genus may have only a single species.

At times a family may be raised to superfamily rank — the sub-
families then becoming families. Thus the families Ixodidae and
Argasidae belong to the superfamily Ixodoidea. The termination for a
superfamily is "oidea".

When there are a number of families agreeing closely in some striking character-
istic, we group them together into an Order; thus, the family of mosquitoes closely
resembling many other families of insects in possessing a pair of well-developed
wings are grouped in the order Diptera; all of which resemble certain other animals
in the possession of a distinct head, thorax and abdomen with three pairs of legs
projecting from the thorax. This collection of animals we call a Class; thus, we
speak of the class Insecta. It will be observed that the insects have no internal
skeleton, but instead a chitinous cuticle, the exoskeleton. Spiders, ticks, etc.,
resemble them in this respect, and we now apply to all such animals the wider
designation, Branch or Phylum Arthropoda.

Inasmuch as the animal kingdom is divided into the branches Protozoa, Pori-
fera, Ccelenterata, Echinodermata, Vermes, Arthropoda, Mollusca and Chordata,
we see that the branch is the largest grouping we employ. To descend in the
scale we have belonging to the branch, the classes; to the class, the orders; to
the order, the families; to the family, the subfamilies; to the subfamily, the
genera; to the genus, the species. Occasionally a species is further divided into
subspecies.

By a type species we understand the species of a genus always re-
ferred to as representing the genus.

While other species of a genus may for good reason be transferred to another
genus the type species is permanently in the genus. Many favor alliteration for
type species, as Heterophyes heterophyes. When a species is transferred to a new
genus the specific name goes with it.

The male animal is designated by the sign of Mars (d"), the female by that of
Venus (9).

KEY TO ANIMAL PARASITES

245

CLASSIFICATION OF PHYLA OF IMPORTANCE IN ANIMAL PARASITOLOGY
(According to Stiles)

1. Unicellular animals, as the parasites of malaria Protozoa.

Pluricellular animals; metazoa 2

2. Body more or less flattened dorsoventrally 4

Body ordinarily round in transverse section

3. Body never annulated, never provided with legs; no jaws present 5

Body annulated, or at least provided with mouth parts; usually breathe through

a tracheal system; adults with jointed legs 7

4. Intestine, but no anus, present; one or two suckers present; body not seg-

mented; parasitic in liver, lungs, blood, intestine; occasionally elsewhere;
flukes Trematoda.

Intestine absent; two or four suckers on head; body of adults segmented; tissue
usually contains calcareous corpuscles; adults (tapeworms) parasitic in intes-
tine; larvae (bladder worms) parasitic elsewhere Cestoda.

Intestine and anus present; ventral sucker on posterior end; body annulated like
an earthworm; parasitic in upper air-passages, or externally, leeches, blood-
suckers Hirudinei.

5. Intestine absent; armed rostellum present; very rare in man, in intestine, thorn-

headed worms Acanthocephali.

Intestine present; no armed rostellum 6

6. Intestine rudimentary in adult; rare, accidental parasites in intestine of man, hair

snakes or horse-hair worms Gordiacea.

Intestine present; parasitic in intestine, muscles, lymphatics, etc., very common
and important; roundworms Nematoda.

7. Six legs present in adult; wings present in most species; larvae annulated much like

an earthworm; breathe through tracheae; adults ectoparasites; occasionally
larva is parasitic under skin, or in wounds, or an accidental parasite in the
intestine; insects Insecta.

Eight legs present in adult, six legs in larva; head and abdomen coalesced;
ectoparasites; some burrow under the skin or live in the hair follicles;
acarines Acarina.

Four claws around the mouth; larva encysted in various organs; adult occasion-
ally parasitic in nasal passages; tongue worms Linguatulidae.

Numerous legs present; occasionally accidental parasites in nasal passages or
intestine, thousand leggers Myriapoda.

There are certain terms employed in animal parasitology which it is
necessary to understand. Among these we shall refer to the following:

1. True Parasitism. — By this is understood the condition where the parasite does
harm to the host, deriving all the benefit of the association. A good example of this
would be the hookworm infecting man or animals.

2. Mutualism. — In such an association there is mutual benefit to each party of the
association. An instance of this would be the presence of colon bacilli in the intes-
tines. The bacillus is furnished a suitable habitat and in return protects its host
against strictly pathogenic bacteria.

246 CONSIDERATIONS OF CLASSIFICATION AND METHODS

Another example would be the oyster crab found inside the oyster shell.

3. Commensalism. — Here there is benefit to the parasite, but no injury to the
host. An example of this kind would be furnished in the case of the Trichomonas
vaginalis which lives in the vaginal mucus, but so far as known, does no injury to the
host.

If the Entamceba coll be nonpathogenic this would be another example.

4. Nomenclature. — When the thousands of different species, genera,
etc., of animals are considered, it will be readily perceived that, unless
some system existed for their designation, indescribable confusion
would prevail. To avoid this, the International Code, based on the
rules of Linnaeus (tenth edition of Systema naturae, 1758, is basis of
binary zoological nomenclature) , requires Latin or Latinized names.

In printed matter the zoological name should be in italics, that of the family in
Roman type. The name of the author of a specific name is written immediately after
the name without punctuation and may be followed by the year of publication set off
by a comma, thus: Ascarls lumbricoldes Linnaeus, 1758. Should the name of the
author appear in parentheses it indicates that he proposed the specific name but
placed the species in another genus than that in which it now appears, and the name
of the author responsible for placing the species in the present genus may be written
after the name of the original author of the species; for example, Davainea mada-
gascariensis (Davaine, 1869) Blanchard, 1891, tells us that Davaine proposed the
specific name madagascarlensls in 1869 but placed it in some other genus and that
Blanchard in 1891 transferred it to the genus Davainea. There are certain rules
governing the naming of animals. Of these, the law of priority provides that the oldest
published name, under the code, of any genus or species is its proper zoological name.
The history of the naming of the organism of syphilis illustrates this well. Schau-
dinn gave this organism in 1905 the name of Splroch&ta pallida. Ehrenburg, in
1838, had used the name Splrochata for animals of a different character, so that this
designation of the genus was not permissible under the code. Villemin, a little later,
proposed the generic name Splronema. This term, however, was found to have
been used in 1864 by Meek for a genus of molluscs and by Klebs in 1892 for a genus
of flagellates. Consequently, being a homonym, it was not available.

(A generic name can be applied to only one animal genus and if a similar name is
subsequently given another genus it is a homonym and is to be rejected.)

On December 2, 1905 Stiles and Pfender then proposed the name Micros pironema,
but as Schaudinn published on Oct. 26, 1905 the designation Treponema, the
name Treponema pallldum had to be accepted as the proper zoological name for the
organism of syphilis.

Of unusual interest is the question of the name of the old-world hookworm.
Dubini, in 1843, named a nematode found by him in man Agchylostoma. By the
law of priority this spelling would have been the correct one had he not stated in a
footnote that the generic name was derived from two Greek words cryxuXoer and
or6/Lta. Having indicated the origin of the name it became subject to the rules for
correct transliteration, which is Ancylostoma.

ZOOLOGICAL NOMENCLATURE 247

In case of larva and adult or male and female, formerly considered
different animals but subsequently found to be the same, the oldest
available name becomes the name of the species.

Another point is that names are not definitions, consequently the fact of lack of
appropriateness of any name is no objection to its continuation. This will appeal
to anyone as a wise provision, because if a different name were substituted each time
a designation more descriptive or applicable was invented it would be utterly destruc-
tive to system. When it is considered that some of our parasites have approximately
fifty different designations, for the most part given by medical observers, it will be
appreciated how much the zoologist has aided us in trying to eliminate all but the
single proper zoological name.

It is a rule of zoological nomenclature that zoological names are
independent of botanical ones so that the prior use of a generic name
for a plant is not an objection to its use for an animal.

The objections so frequently heard among physicians in connection with adopting
new names for old ones are not well founded. Wherever confusion has reigned, the
establishment of order always results in temporary greater confusion. There
is no doubt that the student taking up this subject a few years hence will have the
satisfaction, thanks to the zoologist, of only having to burden his mind with one name
for one parasite.

There is only one correct 'name for an animal and all other names
are synonyms.

The principal cause of changes of names is that our conception of the relationships
of animals changes.

5. Terminology. — This applies to appropriate designations for different organs,
symptoms, etc., and is not subject to any rule other than that of good usage.

Thus the terms cirrus in the case of the male copulatory organ of flukes, spicule
for the same in nematodes and penis in connection with insects would be instances
of terminology.

6. Pseudoparasitism. — Where organisms enter the body accidentally and when
such sojourn in the body of man plays no part in the life history of the organism
we employ the term pseudoparasitism. For example: Fly larvae swallowed by
man and passed out in the faeces. We also use the terms temporary parasites
(bedbug) and permanent parasites (liver fluke).

7. Hosts. — The animal in which a parasite undergoes its sexual life is called the
definitive or final host, that in which it passes its larval existence the intermediary
host. For example: Man is the intermediary host of the malarial parasite, the
mosquito the definitive host. A single animal may, however, be both definitive
and intermediary host; thus Trichinella may pass its larval existence in the muscles
of man and its sexual life in his intestines.

8. Heredity, Congenitalism.— Hereditary characteristics are those which were
present in the ovum or spermatozoon before fertilization; congenital ones those
which occur after fertilization. South African tick fever is probably an instance

248 CONSIDERATIONS OF CLASSIFICATION AND METHODS

of heredity, the spirochaetes having been found in the ovary and ova of the fema
tick.

9. Heterogenesis, Parthenogenesis. — Offspring differs from parent, but after
one or more generations there is reversion to the parent form.

Strictly speaking the term heterogony applies to reproduction when a sexual
generation alternates with a parthenogenetic one. Where a nonsexual genera-
tion, as by division or budding, alternates with a sexual one the process is called
metagenesis. In parthenogenesis reproduction eggs develop without the occurrence
of fertilization by spermatozoa.

In coccidiosis we have a sexual cycle (sporogony) alternating with a nonsexual
one (schizogony) . In the infection with Strongyloides we have a sexual cycle
alternating with a parthenogenetic one. In malaria we have a sexual generation,
a nonsexual one and according to Schaudinn, a parthenogenetic one, which latter
accounts for malarial relapses.

10. Homology and Analogy. — By homology we understand the anatomical
correspondence of the organ of one animal to that of another. Thus the foreleg of
a quadruped and the wing of a bird are homologous organs. Analogy refers to
physiological or functional agreement, thus the lungs of mammals and gills of fish,
both with respiratory functions, are analogous organs. The first trace or appear-
ance of an organ in an embryo is known as the anlage of the organ.

11. Protista. — Haeckel proposed this name for unicellular animals and plants,
thus including protozoans and protophytes in a kingdom separate from the animal
and vegetable kingdoms.

We have sufficient difficulty in drawing the line between an animal and vegetable
organism so that to make a demarcation of a new kingdom from the two usually
recognized would add to our difficulties.

nale

CHAPTER XVI

Class

THE PROTOZOA

CLASSIFICATION OF PROTOZOA
Order Genus

Rhizopoda

(Sarcodina)
These throw out protoplas-
mic projections called pseudo-
podia.

Gymnamceba

Flagellata

(Mastigophora)
These move by means of
undulating membranes or
flagella.

Infusoria

(Ciliata)

These have contractile vacu-
oles and numerous fine cilia
which are shorter than flagella
and have a sweeping stroke.

Sporozoa

These have no motile organs.
They live parasitically in the
cells or tissues of other animals.
Reproduction by spores.

Entamoeba

Leydenia

Spirochaeta

Schizotrypanum
Treponema

Trypanosoma

Trichomonas

Tetramitus

Lamblia

Babesia

Leishmania

Heterotricha Balantidium

Coccidiaria

Eimeria
Isospora

Haemosporidia Plasmodium

249

Species
E. coli

E. histolytica
E. tetragena
E. gingivalis

L. gemmipara

S. recurrentis
S. vincenti
{ S. duttoni
S. carter!
S. refringens
S. cruzi
T. pallidum
T. pertenue
T. gambiense
T. rhodesiense
T. vaginalis
T. intestinalis
T. mesnili.
L. intestinalis
B. bigemina
L. donovani
L. tropica
L. infantum
B. coli

E. stiedae
I. bigemina

P. vivax
P. malariae
P. falciparum

250 THE PROTOZOA

NOTE. — Hartmann and others have grouped the Haemosporozoa and the Haemo-
flagellata in an order BINUCLEATA. The main characteristic is the possession of
two differentiated nuclei, the kinetonucleus and the trophonucleus, at some develop-
mental or transitional stage. While trypanosomes plainly show these characteristics
certain others, as the malarial parasites and the leishman-donovan bodies, having
been modified as the result of cell parasitism, do not do so. This grouping together
of the blood flagellates and sporozoa under the name Binucleata has been considered
by many protozoologists as possibly convenient but not resting on sufficient ground
to cause organisms with similar life histories as Plasmodium and Coccidium to be
separated and the former to be placed with the blood flagellates in a new grouping.

THE PROTOZOA

By the term protozoa we understand a branch of animals in which a
single cell is morphologically and functionally complete; it is not one of
a number of cells going to make up a complex individual and dependent
on such a combination as is the case with the metazoa (there is no
differentiation into tissues in protozoa).

Recognizing the fact that certain protozoa have characteristics which make
it impossible to draw a distinction between them and plants Haeckel has proposed the
name Protista as a designation for all simple and primitive living organisms whether
they be plants or animals. In such a classification we would have the kingdom of
Protista as well as the animal and vegetable kingdoms. In such a grouping the bac-
teria would be the lower types and the fungi and protozoal organisms the higher ones.

The protozoal cells are made up of protoplasm which is divided into nucleus and
cytoplasm. The cytoplasm is at times separated into an external, hyaline portion,
the ectoplasm or ectosarc and an internal granular portion, the endoplasm or endo-
sarc. The functions of the ectosarc are protective, locomotor, excretory and sensory;
those of the endosarc trophic and reproductive. Protozoa may be holozoic (animal
like) or holophytic (plant like), saprophytic (fungus like), or parasitic (living at the
expense of some other animal or plant).

The nucleus is characterized by concentration of the so-called chromatin sub-
stance of the cell. This chromatin however is usually combined with achromatin.
The usually accepted test for chromatin is the staining affinity for basic aniline dyes.
This test is now known to be unsatisfactory as other substances than chromatin may
stain even more intensely. When chromatin is scattered through the cytoplasm, as
extranuclear aggregations, such chromatin granules are called chromidia. There are
cells where the chromidia take the place of the nucleus and from which a nucleus
may be formed. Chromidia may arise from nuclei and nuclei from chromidia. The
nucleus is made up of a network of linin in which achromatic reticulum is contained
the nuclear sap or karyolymph. As a rule an achromatic nuclear membrane, con-
tinuous with the reticulum, separates the nucleus from the cytoplasm. In addition
we have a substance which is achromatic (plastin) and which is the imbedding sub-
stance for chromatin grains. These plastin chromatin combinations are called
karyosomes. The nucleoli are probably pure plastin. Plastin is to be regarded as a

RHIZOPODS 251

secretion or modification of chro matin made to serve as a matrix for the chromatin.
Chromatin may be concentrated in a single mass so that the nuclear space looks
like a vesicle with a central chromatin mass (vesicular nucleus) or numerous chro-
matin grains may be scattered through the nuclear space (granular nucleus). The
centrosome, which presides over cell division, is usually located just outside the
nucleus. In some protozoa however the centrosome is within the nucleus and
is often seen inside of a karyosome and is then called a centriole. The centrosome
may also function over kinetic activities (flagellar motion) and is then termed
blepharoplast.

When appearing as a small granule at the base of the flagellar apparatus it is called
the basal granule. When there are extensions from it to the nucleus we have
rhizoplasts.

Certain protozoa, as trypanosomes, show a differentiation of nuclei, the larger
trophonucleus governing the functions of general metabolism and the smaller kine-
tonucleus directing the motor activities. Infusoria have a larger macronucleus
which contains vegetative chromatin and a smaller micronucleus which contains
reserve reproductive chromatin.

Reproduction of protozoa may be by fission, when the nucleus and cytoplasm
divide into two by simple division.

When the nuclei divide into a number of daughter nuclei, which is followed by
multiple division of the cytoplasm, we have sporulation.

Instead of fission we may have sexual reproduction or conjugation (zygosis).
Here the nuclei of the separate sexual individuals (gametes) are termed pronuclei and
the product of their fusion a synkaryon.

Where a single cell has division of its nucleus with subsequent fusion of these
daughter nuclei to form a synkaryon the process is termed autogamy.

If two similar cells conjugate the term is isogamy; if dissimilar as the macroga-
metes and microgametes of malaria, anisogamy.

The process of sexual union is termed syngamy and is of two kinds (i) when the
two gametes fuse completely or copulation and (2) when they remain separate
and only exchange nuclear material or conjugation.

The structures of protozoa concerned in movement, metabolism, etc., are termed
organelles. Of the former, pseudopodia, flagella, cilia and myonemes (contractile
fibrils which give support to the body cell of certain protozoa) may be given and food
vacuoles and contractile vacuoles of the latter. The contractile vacuole which is
probably an excretory organelle is absent in almost all parasitic protozoa. It is
however present in ciliates.

RHIZOPODA (SARCODINA)

In this class of protozoa the pseudopodia serve the double purpose
of nutrition and locomotion. These protoplasmic extensions may be
quite broad or very narrow — the lobose and the reticulose.

As a rule, the thicker the pseudopod the more rapid the movement. Some rhizo-
pods have hard shell-like coverings which are secreted in or on the ectosarc. These
skeletons have openings through which the pseudopods project. The pseudopodia
may be made up only of ectoplasm or both ectoplasm and endoplasm may take part.

252 THE PROTOZOA

Amoeboid movement always starts in the ectoplasm. In addition to the nucleus,
which the so-called chromatin-staining methods of Romanowsky bring out as reddish
areas, or black with iron haematoxylin, we frequently observe aggregations of chro-
matin-staining material in the cytoplasm. These cytoplasmic chromatin bodies
(chromidial bodies) are of importance in differentiating the encysted pathogenic
amoeba from the nonpathogenic one. This extranuclear chromatin is supposed to
play a part in the more intricate divisions which such protozoa undergo. Food
vacuoles and contractile vacuoles are present in many rhizopods.

INTESTINAL AMCEB^E

There are certainly two species of intestinal amoebae having man
for a host, the one pathogenic, Entamceba histolytica and the other a
harmless commensal, Entamoeba coli.

Schaudinn, in 1903, described the pathogenic amoeba, which he named E. histolyt-
ica, as follows: i. Distinct, highly refractile and tenacious ectoplasm. He consid-
ered this tough external portion of the cytoplasm as the explanation of the ability
of the pathogenic amoeba to bore its way into the intestinal submucosa. 2. Eccen-
tric nucleus which was indistinct by reason of little chromatin. 3. Reproduction by
peripheral budding in which small aggregations of chromatin reached the periphery
of the cytoplasm and, enclosed in a resistant capsule, broke off from the parent
amoeba and constituted the infecting stage.

For the nonpathogenic E. coli he noted, i. No distinction between a granular
endoplasm and refractile ectoplasm. 2. Centrally placed and sharply outlined
nucleus, rich in chromatin and 3. Encystment with the formation of eight nuclei,
which nuclei or amcebulae form the infecting stage.

The pseudopodia of E. histolytica are actively projected as long finger-like processes
which show the ectoplasm quite distinctly, while the pseudopodia of E. coli, are
lobose and sluggishly projected and show a uniformly opaque grayish color.

In 1907 Viereck and later Hartmann recognized a pathogenic
amoeba with four nuclei in its encysted form, to which was given the
name E. tetragena.

All authorities now consider that Schaudinn made an error in observation as to
the existence of peripheral budding for E. histolytica, so that we recognize but two
types of encystment, one with a larger cyst and thicker cyst wall, with eight nuclei
and an absence of chromidial bodies — E. coli and the other, smaller, with a thin cyst
wall, four nuclei and chromidial bodies in the encysted stage, the pathogenic amoeba,
E. histolytica. Synonym. E. tetragena.

In the vegetative stage the human amoebae are best differentiated by the nuclear
structure. In E. coli the nucleus is vesicular with a thick nuclear membrane and
the chromatin chiefly deposited on the undersurface of the nuclear membrane.
In haematoxylin-stained specimens this chromatin often seems deposited in quadrant
aggregations.

Whitmore noted a broad clear zone as surrounding the karyosome of the tetra-

THE AMCEB.E OF MAN

253

gena nucleus of E. histolytica, a distinction from the central karyosome of E. coll
which has only a very narrow clear zone surrounding it.

At the present we differentiate between two types of nucleus— the
histolytica one, which is found in acute attacks of dysentery and

0

FIG. 59. — Important pathogenic protozoa of the' intestinal tract. (la) Motile E,
coli. Note large amount and peripheral arrangement of chromatin in nucleus. ( ib )
Encysted E. coli. Note larger size than E. histolytica cyst, 8 ring form nuclei and
absence of chromidial bodies. (2) Motile E. histolytica from acute dysenteric stool.
Note histolytica nucleus with scanty chromatin. (3) Tetragena type of E. histo-
lytica from case of chronic dysentery. Note greater amount of chromatin and central
karyosome with centriole. (4a) Preencysted E. histolytica from carrier. Note small
size and heavy peripheral ring of chromatin in nucleus making this feature of chrom-
atin in nucleus similar to the larger E. coli. (4_b) Encysted E. histolytica from
dysentery convalescent. Note small size, 4 ring nuclei and a dark chromatin stain-
ing mass, "chromidial body." (sa and 5b) Motile and encysted cultural amoebae
from Manila water supply. (6a and 6b) Oocyst and sporozoite production in 4
spores of Eimeria stiedae. (ya and 76) Oocyst with 2 sporoblasts and oocyst with
2 spores containing 4 sporozoites of Isopora bigemina. (8a and 8b) Vegetative and
encysted Trichomonas intestinalis . (pa and pb) Vegetative and encysted Lamblia
intestinalis. (10) Balantidium coli. Illustrations of amoebae from Walker — others
from Doflein.

shows a delicate nuclear membrane with scattered chromatin granules
in its inner side and as a rule a very small central karyosome. The
tetragena type has a much thicker nuclear membrane and is the one
generally found in pathogenic amoebae in chronic dysentery. Of

254 THE PROTOZOA

not;

course instead of the vegetative nucleus of tetragena type one may
in chronic cases or in convalescence come across the four nuclei cysts
of E. tetragena.

Animal experimentation upon kittens with E. coli by Schaudinn, Craig and
Wenyon have been unsuccessful as to production of dysenteric manifestations. On
the other hand all of these experimenters produced typical lesions and dysenteric
manifestations in kittens injected rectally or fed with material containing patho-
genic amoebae.

Wenyon produced a liver abscess in one of his experiments. In man the dislodg-
ment of amoebae containing material from amoebic intestinal ulcerations and the
plugging of the portal capillaries by such emboli gives us the starting-point of a liver
abscess. The exciting cause is Entamceba histolytica which in the liver continues
the same production of a gelatinous necrosis as is carried on in the submucosa of the
large intestine or appendix.

Darling has been so successful in his experimental work with kittens that he
compares the colon of a kitten to a test-tube and suggests the procedure of rectal
injections of material containing amoebae as a means of differentiating the two human
amoebae.

On the other hand Walker was unable to infect kittens and monkeys with material
containing pathogenic amoebae and he makes the statement that such failures would
indicate the greater susceptibility of man to infection, as he was able to infect 17
out of 20 men with one feeding of such material.

Recently Walker and Sellards have published a most important
paper.

The experiments were made in men who had been under observation for years
at Bilibid Prison, whose food was cooked and the water they drank distilled. More-
over, there were complete records of examination for intestinal parasites, including
entamcebae. They were under complete control and the existence or possibility of
natural infection with amoebae was reduced to a minimum. All the men fed patho-
genic amoebae were volunteers and each signed, in his native dialect, an agreement to
the conditions of the experiment.

The first series of experiments was with cultural amoebae, in order to refute state-
ments that amoebae cultivated from water or other nonparasitic sources, as well
as from dysenteric stools, are capable of living in man parasitically or of producing
dysenteric symptoms. Twenty feeding experiments were made by Walker and
Sellards with cultures of amoebae without the development in a single instance of
dysentery or the finding of such amoebae in the stools upon microscopical examina-
tion. In 13 cases they recovered the amoebae in cultures from the feces from the
first to the sixth day, but never afterward. They stated definitely that cultural
amoebae are nonpathogenic.

The next experiments were with Entamoeba coli. In the 20 cases fed with material
containing Entamaba coli there was a uniform failure to recover them culturally and
in no instance was dysentery produced. Seventeen became parasitized as the result
of a single feeding in from one to eleven days, the entamcebae being found in the
stools and persisting in their appearance in the stools for extended periods. They

EPIDEMIOLOGY OF AMCEBIASIS 255

concluded that Entamosba coli is an obligate parasite, nonpathogenic, and cannot be
cultured.

The third series of 20 feedings, carried on by Walker alone, was with Entamceba
histolytica. The material was mixed with powdered starch or magnesium oxide and
given in gelatin capsules. In these experiments they obtained tetragena cysts in
the stools of men fed only motile Entamceba histolytica, and motile Entamosba histolyt-
ica in the stools of men who were fed only tetragena cysts and, finally, an alterna-
tion of motile E. histolytica and tetragena cysts in the stools of a man having a
recurrent attack of amoebic dysentery.

Seventeen of the men became parasitized after the first feeding; one required three
feedings, and two, who did not become parasitized at the first feeding, were held as
controls. The average time for parasitization was nine days. Only four of the 18
parasitized men developed dysentery, which came on after 20, 57, 87, and 95 days,
respectively, after the ingestion of the infecting material.

In four cases fed with material from acute dysenteric stools or from amoebae con-
taining pus from liver abscess, and containing motile amcebae, there was no resulting
dysentery, the four cases of experimental dysentery resulting from feeding of material
from normal stools of carriers.

As regards the cases which became parasitized, but did not develop dysentery,
it is suggested that the amoebae live as commensals in the intestine of the host and
only penetrate the intestinal mucosa and become tissue parasites when there occurs
depression of the natural resistance of the host or as the result of some lesion of the
intestine. That the pathogenic amcebae are more than harmless commensals, how-
ever, is shown by the fact that they alone, and not the nonpathogenic Entamosba
coli, are capable of penetrating a possibly damaged intestinal mucosa.

Epidemiology. — The old idea that water, fruit or vegetables, from
which one can isolate amcebae upon culture, are sources of infection
must be abandoned, as such cultural amcebae are known to have no
pathogenic relation to man.

The chief factor in the spread of amoebic dysentery would seem to be the encysted
amcebse in the stools of convalescents or healthy carriers rather than the motile ones
in dysenteric stools. This probably explains the endemic rather than epidemic
characteristics of the spread of amoebic dysentery because if the innumerable vege-
tative amcebse in dysenteric stools were equally operative with the more sparsely
eliminated cysts there would be epidemics of amoebic dysentery similar to those of
bacillary dysentery.

Our present view is that the carrier is the chief factor in the spread
of amoebic dysentery and when such an individual has to do with the
preparation of food he becomes a particular source of danger.

Vegetative amcebze undergo disintegration in a short time after the stool is passed
so that they are probably rarely concerned in amoebic infections but the resisting
cysts may be washed from a dried stool into a water supply or even be transported in
dust to lodge on unprotected food stuffs.

256 THE PROTOZOA

Flies may possibly act as transmitting agents.

We can as a rule differentiate bacillary from amoebic dysentery by the more
sudden and acute onset of the former together with fever and other evidences of
toxaemia.

Again the number of stools in bacillary dysentery is usually greater and the amount
of each stool less in quantity. The stool of bacillary dysentery is of a milky white-
ness from the large number of pus cells, while that of amoebic dysentery is more
viscid and tinged with disintegrated blood giving it a grayish green or brown color.
The mucopurulent mass in bacillary dysentery may be flecked or streaked with
blood. The therapeutic results following emetine injections are of value in diagnosis.
Gangrenous types of dysentery are similar whether due to bacillary or amoebic
infection. Chronic dysentery of bacillary origin is much like amoebic dysentery
clinically.

Laboratory Diagnosis. — In the fresh specimen of the milky muco-
purulent mass of bacillary dysentery one observes large numbers of
pus cells and particularly very large phagocytic cells which greatly
resemble amoebae. Upon staining with Gram's stain one may find
numerous Gram-negative bacilli in the cystoplasm of the cell.

The large cells which resemble amoebae are often vacuolated, thus intensifying
the similarity. They are nonmotile, however, and do not show the small ring
nucleus which is so characteristic of the vegetative human amoebae. The nucleus
of the confusing cells is also larger, approximating one-fourth the size of the cell.

For bringing out the nuclear characteristics of human amoebae Walker recom-
mends fixation of thin moist smears in sublimate alcohol (absolute alcohol i part,
sat. aq. sol. bichloride 2 parts) for ten to fifteen minutes. These smears are then
well washed with water and stained with alum haematoxylin for five minutes. The
nuclear characteristics are noted under etiology. With vegetative amoebae I have
obtained beautiful results with vital staining which can best be done by tinging the
faeces emulsion with a i% aqueous solution of neutral red. I have also had good
results by emulsifying the faeces in a drop of i or 2% formalin and then adding a
drop of 2% acetic acid. The mixture is then tinged with either neutral red or
methyl green.

For distinguishing the encysted form of Entamceba coli one can obtain beautiful
results by emulsifying the faeces in Gram's iodine solution. Owing to the glycogenic
reaction given by E. coli, the round amoebae, with its eight nuclei stands out
very distinctly.

For diagnosing the four nucleated cyst of the pathogenic amoeba one
gets better results with haematoxylin as this brings out not only the four
nuclei but the chromidial bodies as well. The use of the iodine solu-
tion is, however, almost as satisfactory for pathogenic cysts as for
nonpathogenic ones. Again where cysts are not abundant one has
little success in finding them in iron haematoxylin preparations which

DIFFERENTIATION OF AMCEBAE

257

staining I prefer to alum haematoxylin. It was formerly customary
to recommend the administration of salts prior to examining for
amoebae.

Walker warns that such a procedure gives us amoebae which are difficult to differ-
entiate, the nuclear characteristics of E. coli and the tetragena nucleus of E. histo-
lytica being much alike as they both contain much chromatin. In a dysenteric
stool the histolytica type of nucleus, containing but little chromatin, does not
resemble the nucleus of E. coli.

He prefers the examination of formed stools obtained without a
purgative.

Walker also notes the advantages of examining a specimen with a %-inch ob-
jective as encysted amoebae are easily picked up. In opposition to the usual recom-
mendation of text-books to report only on motile amcebae, he recommends the
making of a differential diagnosis on nonmotile encysted forms. This however is
now generally accepted by experienced workers as true. The four nuclei cysts of
E. histolytica are from n to 14 microns in diameter while the eight nuclei ones of E.
coli are from 1 6 to 25 microns in diameter.

Sellards and Baetjer note that inoculation of kittens per rectum or
by feeding dysenteric stools rich in amcebae has resulted in infection
in about 50% of experiments.

By inoculating the material directly into the caecum they were
able to infect every one of their kittens. They were also able to
propagate a strain of amcebae through a series of animals for several
months.

The intracaecal inoculations yielded positive results in diagnosis of
human amcebiasis when the clinical manifestations were obscure
and the amcebae in the discharges so few and atypical as to make such
an examination unsatisfactory.

As differentiating the two entamcebae Walker gives the following table:

MOTILE STAGE
A. Entamceba histolvtica

B. Entamceba coli

1. Appearance hyaline.

2. Refractiveness more feeble.

3. Movements active in the fresh stool.

4. Nucleus more or less indistinct.

5. Chromatin of nucleus scanty.

17

1. Appearance porcelaneous.

2. Refractiveness more pronounced.

3. Movements sluggish.

4. Nucleus distinct.

5. Chromatin of nucleus abundant.

258

THE PROTOZOA

ENCYSTED STAGE
A. Entamceba histolytica

B. Entamoeba coli

1. Cyst smaller.

2. Cyst less refractive.

3. Cyst usually contains elongated re-
fractive bodies known as "chromidial
bodies."

4. Nuclei never more than four.

5. Cyst wall thinner.

1. Cyst larger.

2. Cyst more refractive.

3. Cysts do not contain "chromidial
bodies."

4. Nuclei eight, occasionally more.

5. Cyst wall thicker.

A method for bringing out the nuclear features is as follows: take a loopful of
2% acetic acid and a loopful of 2% formalin. Tinge the mixture to a rose color
with neutral red and then stir in a little saturated aqueous solution methyl green,
using a toothpick which has been dipped into the methyl green.

In staining with iron haematoxylin or better with phosphotungstic haematoxylin
proper fixation is very important. Fix in 100 parts of sat. aq. sol. bichloride to
which is added 50 c.c. absolute alcohol and 5 drops glacial acetic acid. The stain
should be poured on the moist smear of faeces. The fixative should be heated to
6o°C. and should only act for ten to twenty seconds. Then place in cold sub-
limate alcohol for ten minutes wash in 70% alcohol colored to a rich port wine
color with iodine, then in 70% alcohol, then in water and then stain as preferred.
Some like a carmine stain. The smears should be moist when fixed with bichloride
fixatives. A small loopful of Meyer's albumin fixative to emulsify is a great aid.

AMCEB.E or PYORRHOEA ALVEOLARIS

Recently much importance has been attached to certain amoebae
found deep in the pus pockets of affected teeth. They are best ob-
tained after wiping away the superficial pus and then scraping material
from the depths of the pockets with a wooden toothpick. They may
be emulsified in salt solution but saliva is better for the obtaining of
motility. The name is Entamceba gingivalis and it is probably the one
described under the names E. buccalis and E. dentalis. It rather re-
sembles E. histolytica in having a greenish refractile ectosarc and an
indistinct nucleus. Encysted forms have not been observed.

It is open to question whether these amoebae are the cause or whether various
streptococci bring about the condition. A symbiotic relationship may be opera-
tive. Emetine seems often of value especially when combined with an autogenous
vaccine. The recent enthusiasm for emetine in treatment of pyorrhoea seems to be
disappearing.

Entamceba gingivalis varies from 10 to 25/1 in diameter. The nucleus is much
smaller than those of the intestinal amoebae and has a distinct nuclear membrane en-
closing a deeply stained karysome. On the whole it is poor in chromatin, in this
respect rather resembling the histolytica nucleus.

SPIROCH^TES 259

Pseudopod projection is less active than that of E. histolytica but more so than E.
coli. There is no distinction between endoplasm and ectoplasm and the nucleus is
indistinct. Encysted forms are very rarely seen and these do not show evidence of
reproduction.

FLAGELLATA (MASTIGOPHORA)

In this class of protozoa the adults have flagella for the purposes of
locomotion and the obtaining of food.

Some flagellates more or less resemble rhizopods in being amoeboid and in having
an ectoplasm and an endoplasm. The body is frequently covered by a cuticle
(periplast). Some flagellates have a definite mouth part, the cytostome, which leads
to a blind oesophagus; others absorb food directly through the body wall. In addi-
tion to flagella, some flagellates possess an undulating membrane. All flagellates
possess a nucleus and some have contractile vacuoles. The flagellum may arise
directly from the nucleus or from a small kinetic nucleus, the blepharoplast (micro-
nucleus or basal granule).

The most important flagellates of man are the hsemoflagellates. Among these
we may include the blood spirochsetes and the organism of syphilis, which have
many resemblances to the spiral forms of bacteria, together with the three genera in
which protozoal characteristics are marked, namely, Leishmania, Trypanosoma
and Trypanoplasma. In addition we have flagellates in the intestinal canal and in
the vaginal secretion. Some authors place the genus Piroplasma with the flagellates
and there has been controversy concerning the nature of certain projections from
these bodies. It would seem preferable, however, to consider them under the
Sporozoa.

Spirochaeta

The generic term Spirochata is applied to flagellates having a spiral
shape, an undulating membrane, and no flagella. This genus is one
about which there are two views: one, that the members belong to the
bacteria; the other, that they are protozoa. The absence of demon-
strable nucleus and blepharoplast makes them apparently vegetable in
nature while the variations in thickness, the fact of transmission by an
arthropod, and indications of a longitudinal, rather than a transverse
division, would indicate protozoal affinities.

It would seem from recent investigations that both methods occur— longitudinal
division occurring when there are few organisms in the blood and transverse at the
height of the infection.

Minchin has adopted the name Spiroschaudinnia, proposed by Sambon, for the
parasitic blood spirochaetes.

Spirochsetes of Relapsing Fevers.— Relapsing fevers are caused by
organisms generally considered as protozoal in their nature and be-
longing to the flagellates.

260 THE PROTOZOA

The generic name Spiroschaudinnia is preferred by some to the more commonly
accepted Spirochata. East and West African relapsing fevers, or tick fever, is
caused by S. duttoni and the transmission is through the bite of an argasine tick,
Ornithodorus moubata. Not only does the tick itself become infected by the taking in
of blood containing spirochaetes but likewise transmits the infection to its progeny.
Leishman considers that when the spirochaetes are taken into the alimentary tract
of the tick there is a breaking up of the spirochaetes into small granules which reach
the Malpighian tubules. They also invade the ovary and the ova. It was thought
that these granules were the infecting agents and that they were excreted in the
fluid of the coxal glands or passed out with the faeces. More recently it has been
claimed that these granules have no relation to the infection, which is due to spiro-
chaetes as such. At any rate this infection of man seems to be by the contamination
method, the material from faeces and coxal glands being rubbed into the wound made
by the tick bite. The ticks hide in the cracks about the old native huts and bite the
sleeping inmates. There may be quite a local reaction at the site of the bite.

nly

FIG. 60. — Spirochaetae of relapsing fever from blood of a man. (Kolle and

Wassermann.)

Spircchata duttoni has been cultured by Noguchi, by utilizing his methods for
culturing the organism of syphilis. In such cultures he has noted longitudinal
division rather than transverse, this fact rather favoring a protozoal as against a
bacterial nature. This spirochaete is from 24-30 microns long, about 0.45 microns
broad and has a corkscrew motility. It is readily transmissible to a number of
laboratory animals, as white rats, etc. The spirochaete of northern African re-
lapsing fever, S. Berbera causes the disease as seen in north Africa and Egypt. It
is transmitted by lice, Nicolle and others having shown that the spirochaetes make
their way from the alimentary tract to the body cavity of the louse. They have
shown that the bite alone of an infected louse is innocuous and also that the faeces
are noninfective, when injected into monkeys. Emulsions of infected lice, however,
when rubbed into wounds, produce the disease in monkeys.

It is by crushing the louse, by scratching or otherwise, that the
spirochaetes contained in the ccelomic fluid reach and penetrate the

SYPHILIS 26l

wound of the bite. This is therefore a contaminative method of
infection.

Mackie has shown that the Indian relapsing fever, which is caused by S. carteri, is
probably transmitted by the louse, and it is probable that the conditions under which
the infection takes place are similar to those occurring with S. berbera infections.
With the European relapsing fever, bedbugs have been suggested as transmitting
agents. The probabilities however are that this infection is caused by lice.

A relapsing fever of Persia is transmitted by a tick of the genus Ornithodorus.
There is great variation in the description of the different spirochjetes, and fre-
quently measurements are given for short forms and long forms. They also vary
from wave-like lines to corkscrew spirals. Again, different species have different
types and different activities of movement. As a rule they are about 20 X 0.4
microns. Noguchi has recently cultivated the various species of pathogenic human
spirochaetes by employing a method similar to that used in cultivating the organism
of syphilis. He noted longitudinal division in his cultures.

S. vincenti. — This is a very delicate spiral-shaped organism which has been found
in conjunction with a fusiform bacillus in a throat inflammation, usually termed
Vincent's angina.

S. refringens. — This Spirochata is frequently associated with the Treponema
pallidum and is common in genital ulcerations. It is thicker, has less regular and
more flattened curves and stains more readily. By "dark ground illumination" it
is thicker, of a yellow tint instead of pure white, and moves in its entire length.

i

Treponema

The genus Treponema has no undulating membrane and has a
flagellum at each end.

Treponema pallidum (Spirochseta pallida). — This is the cause of
syphilis. It was discovered by Schaudinn and Hoffmann in 1905 in
various syphilitic lesions and was cultured by Noguchi in 1912. It is
characterized by the very geometric regularity of the spirals, which are
deeply cut, and in focusing up and down continue in focus (like a
corkscrew). They require about thirty minutes to stain distinctly
with Giemsa's stain and the attenuated ends or flagella should always
be noted before reporting their presence.

Treponemata are found in the cellular areas surrounding the thickened blood-
vessels and in the coats of the larger arteries. To stain them in section Levaditi's
method is the best.

The India-ink method of Burri is highly recommended. Take one loopful of
secretion from a chancre and deposit it on one end of a slide. Emulsify in this a small
quantity of Gunther and Wagner's ink such as can be obtained by touching the end
of a new spatulate toothpick in the ink. Use the toothpick for mixing. I use the
method of sticking a fine glass pipette into the underlying corium of the chancre and

262 THE PROTOZOA

get serum in that way or by squeezing the chancre afterward. Mix and make a
smear as for blood. When dry examine with the oil immersion objective and the
treponemata will be found to stand out as white spirals against a dark background.
Treponemata often appear as if bent in the middle.

Harrison prefers collargol to india ink. One part of collargol is put in a bottle
with 19 parts of water and well shaken. This shaking is repeated. One loopful
of the suspected serum and one loopful of the collargol suspension are mixed and
smeared out and examined as for the india-ink method.

Cultivation. — T. pallidum has been cultivated anaerobically in horse serum by
Schereschewsky. The cultures contained other organisms. Muhlens, by growing
anaerobically on horse-serum agar (i to 3), claims to have obtained pure cultures.
Animal inoculations with this material were negative, however.

Noguchi has cultivated T. pallidum under strict anaerobic conditions in a medium
of ascitic fluid containing a piece of fresh sterile tissue, preferably placenta. The
growth is faintly hazy and does not have an offensive odor. Spirochceta micro-
dentium shows similar morphology but the cultures have a foul odor. Sp. macro-
dentium is similar culturally but differs morphologically.

Luetin. — When cultures of T. pallidum, grown for one or more weeks in ascitic
fluid agar and ascitic fluid are ground in a mortar, heated to 6o°C. for one hour
then, with the final addition of i% trikresol, we have an emulsion called "luetin."
This extract produces an allergic reaction on the skin of certain syphilitics (luetin
reaction). To carry out the test luetin is introduced intradermally at the insertion
of the left deltoid and a control emulsion of agar media injected in the right arm.
A negative result shows as an erythema without pain or papule formation. Positive
reactions show as papules vesicles or even pustules giving rise to discomfort for
several days. Not only do we have papular and pustular type reactions but also
torpid ones (taking ten days or more to develop). While the control side usually
becomes normal in forty-eight hours yet in latent and tertiary syphilis the control
may show almost as marked a reaction. The term "Umstimmung" is applied to
this susceptibility to trauma of the skin of those having tertiary syphilis. Some
cases of parasyphilitic infections which are negative to the Wassermann test give
a positive luetin reaction. Tertiary yaws cases frequently give a positive luetin
reaction. See comparison of Wassermann and luetin statistics.

Noguchi has recently demonstrated T. pallidum in all layers of the cerebral
cortex except the outermost one in 12 cases out of 70 cases of general paresis
examined.

Diagnosis. — In diagnosis either use the dark ground illuminator or make a thin
smear from the sanious oozing after vigorous friction of the chancre with gauze,
taking up this blood-stained serum on the end of a slide and smearing the surface
of a second slide with the adhering material. It is in most cases more satisfactory
to curet the lesion, in this way obtaining material from the areas of the thickened
arteries. Fontana's silver staining method is an excellent one.

In the diagnosis of cerebrospinal syphilis we use, in addition to the Wassermann
test of the blood, (i) the Nonne-Apelt reaction in which about i c.c. of a saturated
aqueous solution of ammon. sulphate is added to an equal amount of cerebrospinal
fluid. If turbidity or rather opalescence appear immediately, or within three
minutes, the test is positive. We now use a ring test. (2) The counting of the
lymphocytes in the cerebrospinal fluid. A lymphocytosis occurs in cerebrospinal

YAWS

263

syphilis, tabes and general paresis. (3) The Wassermann test, using the cerebro-
spmal fluid instead of blood-serum. (4) The colloidal gold test of Lange These
various examinations of spinal fluid are taken up in detail in chapter on cytodiagnosis
and spinal fluid examinations.

T. pertenue.— An organism of similar morphology was first reported
by Castellani as present in yaws. It is found in smears and sections as
with T. pallidum.

Q

:." ..

8

9

§,

eC

FIG. 61. — Binucleata, (Haemoflagellata and Haemosporozoa). i. Schizotrypanum
cruzij (a) Merozoite just entering r.b.c.; (b) fully developed trypanosome form in
blood; (c) form found in intestine Conor hinus; {d) form in salivary gland of Conor hi-
nus; (e) merocyte from the schizogenous cycle in lungs. 2. Leishmania donovani;
Parasites from spleen smear, free and packed in phagocytic cell; (b) and (c) flagel-
late forms from cultures. 3. Trypanosoma gambiense. 4. Plasmodium vivax; (a)
young schizont; (b) uninfected red cell; (c) red cell, punctate basophilia; (d) merocyte;
(e) macrogamete; (/") adult schizont. 5. Plasmodium malaria; (a) half-grown
schizont showing equatorial band; (b) macrogamete; (c) merocyte; (d) young
schizonts. 6. Plasmodium falciparum; (a) red cell showing multiple infection; (b)
young ring from; (c) crescent; (d) young schizont on periphery of r.b.c. 7. (a)
Treponema pallidum; (b) Spirochceta refringens. 8. Treponema pertenue.

A point of distinction between these spirochaetes is that the T. pallidum is found
in abundance in sections from a chancre about the thickened arteries in the corium,
while in sections from a yaws nodule the T. pertenue is found chiefly in the region
of the interpapillary pegs of the Malpighian layer of the epidermis where they bound
the papillary layer of the corium.

264 THE PROTOZOA

T. pertenue has been cultivated in the same way as T. pallidum and Nichols
infected rabbits by intratesticular injection. A disease of Guam known as gangosa
is possibly connected with a tertiary form of yaws. In persons who have had yaws
a positive Wassermann reaction seems to be given in a higher percentage than is
true for syphilis. Salvarsan is also more specific for yaws than for syphilis.

TRYPANOSOMES OF SLEEPING SICKNESS

The African trypanosomiases follow infection with two species of
trypanosomes; the more virulent type of the disease, occurring in
South Central Africa, being due to Trypanosoma rhodesiense, trans-
mitted by Glossina morsitans and that of less severe type, but of
more general distribution, being due to T. gambiense and transmitted
by Glossina palpalis. The very important Trypanosoma brucei, which
is the devastating agent in the African horse, dog and cattle disease,
nagana, is also transmitted by Glossina morsitans and there exists
the opinion that this trypanosome is identical with T. rhodesiense.

These trypanosomes are blood flagellates and are typical of the Binucleata in
possessing two chromatin staining areas, the larger and more centrally situated
mass being the tropho or macronucleus and the smaller, but more deeply staining one,
the kineto or micronucleus (Blepharoplast). Trypanosomes have a fusiform or
fish-shaped body which stains blue. Near the less pointed, nonflagellated end,
usually called the posterior end, is the deeply stained blepharoplast. Behind this
is a vacuole and, taking origin from this part of the trypanosome, is the flagellum.
This borders an undulating membrane attached to the body and then, carried along
to the other extremity, projects free as a long, whip-like flagellum.

In fresh preparations the body of the trypanosome progresses in the direction of
its flagellated end, although occasionally it will be observed to move in the opposite
direction.

T. gambiense varies much in length and breadth. The normal type, as found in
the blood, varies from 14 to 20 microns, while longer forms, 20 to 24 microns, are
growth ones and, in the longest ones (23 to 33 microns), we have those preparing
to divide longitudinally. The normal short forms are the ones from which the
development takes place in the tsetse fly. In width these flagellates are from 1.5 to
2 microns. The blepharoplast is oval and the nucleus situated about the center.

With T. rhodesiense the nucleus is typically located almost adjacent
to the blepharoplast. As a matter of fact it may require the passage of
this trypanosome through rats to bring out these "posterior nuclear
forms," the nuclear location being at times almost entirely that of
T. gambiense. In addition to the characteristics of nucleus being near
the blepharoplast, this trypanosome is more virulent for laboratory
animals than T. gambiense, agreeing in this respect with the more severe
clinical course in man.

has

SLEEPING SICKNESS 265

When the tsetse fly, Glossina palpalis, feeds on a man in whose peripheral circu-
lation there are normal type trypanosomes we have an accumulation of such forms in
the middle and posterior portions of the gut. From the eighth to the eighteenth day
long, slender forms develop and pass forward into the pro ventriculus. None of the in-
testinal forms can cause infection when injected into animals. These proventricular
types work their way into the salivary ducts and thence into the salivary glands,
where further development takes place. Here we have shorter forms developing,
which are similar in morphology to the normal blood type. It is at this stage that
the fly becomes infective by the passing of these trypanosomes down the salivary
ducts and through the channel in the hypopharynx to the subcutaneous tissues of
the person bitten. High temperatures, 75 to 8s°F., are favorable to development,
while low temperatures, 60 to 7o°F., are inimical to development, but do not kill
the ingested trypanosomes. This explains the long period which at times elapses
before a fly becomes infective. Under favorable conditions a fly becomes infective
in twenty to twenty-four days and remains infective the rest of its life, up to 185 days.
The infection is not transmitted to the pupa. This is an inoculative, cyclical or
indirect type of infection. It is usually considered that a tsetse fly whose proboscis
has just been contaminated with trypanosome blood is capable of transferring the
infection for a few hours. This would be a mechanical or direct method of infection
and such power for infection only lasts for a few hours.

When tsetse flies feed on animals infected with trypanosomes only
from 2 to 6% become infective. Again, it has been shown that where
the wild animals on which tsetse flies feed may show an infection of
from 16 to 50% yet not more than two out of every 1000 tsetse flies,
caught and tried out on susceptible animals, show themselves infective.

Both of the human trypanosomes of Africa have been cultured by using the
N.N.N. medium in which rat's blood was substituted for that of the rabbit. Human
blood will also serve as a substitute. Growth however is not constant.

For the laboratory diagnosis we may use peripheral blood with some
thick film method. The examination of preparations from the per-
ipheral blood is usually very discouraging. Very much better results
(in fact some prefer this method to any other) can be obtained by tak-
ing 10 to 20 c.c. of blood into about 25 c.c. of citrated salt solution,
centrifuging two or three times and examining the sediment of the
third centrifugalization. Button and Todd prefer to centrifuge
citrated blood and to collect the leukocyte layer for examination as is
done in opsonic work.

The English workers usually prefer the gland puncture method, using a sterile
but dry hypodermic needle. Water in the needle distorts both leishman bodies and
trypanosomes.

In the sleeping sickness stage trypanosomes can almost constantly be found in the
cerebrospinal fluid.

266 THE PROTOZOA

Some prefer to inoculate susceptible animals, particularly the guinea-pig or
monkey, with blood or gland juice from the suspected case. A very satisfactory
material is an emulsion from an excised gland which may be inoculated intraperi-
toneally into white rats. The further course, after animal inoculation, is the ex-
amination of the blood of these animals for trypanosomes. Usually at the time the
guinea-pigs die we find numerous trypanosomes.

Other tests are (i) Trypanolysis, when unheated suspected serum and trypano-
somes are incubated together for one hour. Normal serum may occasionally cause
disintegration and treated cases give it in only about 45% of cases. Unfavorable
untreated cases give it in about 80% of cases.

(2) The so-called auto-agglutination test is not of much value. In this the red
cells of the blood of a trypanosomiasis case come together in clumps when one
makes a wet preparation. It is not a rouleaux formation. (3) The attachment test
is made by making a mixture of inactivated serum, leukocytes and trypanosomes
and allowing them to be in contact for twenty minutes. A positive test shows
attachment of the trypanosomes to the leukocytes.

Of the more important trypanosome diseases of animals may be
mentioned:

1. Nagana. Pathogenic for domesticated animals in South Africa. T. brucei.

2. Surra. Pathogenic for horses in India and Philippines. T. evansi.

3. Dourine. Transmitted by coitus in horses. T. equiperdum.

4. Mai de caderas. Affects horses in South America. T. equinum.

A harmless infection, especially in sewer rats, is due to T. levrisi. Transmission of
this rat trypanosomiasis can apparently be brought about through the agency of
both fleas and lice. In the flea there is apparently a developmental cycle of a dura-
tion of one week.

There are many trypanosomes in birds, fish, frogs, etc.

Schizotrypanum cruzi (Trypanosoma cruzi). — In 1909, Chagas re-
ported the finding of a flagellate in the intestines of Conorhinus megistus
or, more properly, Lamus megistus. He was also able to transmit the
flagellate to laboratory animals and could culture it on blood agar.

In investigating the matter of the importance of this flagellate,
Schizotrypanum cruzi, in Minas Geraes, Brazil, where the bug was pres-
ent in great numbers in the cracks of the houses of the poor, he asso-
ciated this flagellate infection, which he at first considered trypano-
somal, with a disease of the children of that section.

The bug is a vicious feeder and, from its biting chiefly about the face, has been
called barbiero or barber by the natives. Both the male and female of Lamus bite
and can transmit the disease and although the parasite is not transmitted hereditarily
the nymph is capable of sucking blood and becoming infected.

It requires several months for the insect to go through the egg, larval and pupal
stage to maturity. Some consider this bug to belong to the genus Triatoma. The
insects may live for more than a year and tend to remain in the same house where

r or

BRAZILIAN TRYPANOSOMIASIS 267

they may have become infected but leave such house if it be abandoned by man
Brumpt thinks that the bedbug may also transmit the disease.

S. Cruzi is found in the blood of children during the acute febrile
stage but at other times in children, and as a rule in adults, it is rarely
present in the peripheral blood. The early blood forms are narrow
and very motile. They increase in size and slacken in motility when
they become about 20 microns long. 5. cruzi is characterized by a
very large blepharoplast. Dividing
forms are never seen in the blood. The \
common site of multiplication is in the ) d£2\ /-
cells of the voluntary muscles and heart \zJr ^^^ CV (T^*
and also in the cells of the central ner- &f~\ \£g})

vous system, adrenals, thyroid and bone

marrow. In these tissues the flagellate

i i e 11 FIG. 62. — Schizotrypanum cruzi

takes on a rounded form and undergoes in blood of child with acute type of

binary division. Continued division Brazilian trypanosomiasis. (Mac-

.-, • p , j n . , Nealfrom Doflein after Chaeas.)

converts the infected cell into a cyst.

It is this process going on in various important structures that ac-
counts for the extreme variation in symptomatology and pathology.

Chagas thinks that the gametes for the cycle in Lamus arise from parasites develop-
ing in the lungs of the vertebrate host. Flagellated parasites enter the lungs, lose the
flagellum and become oval in shape, later on dividing into 8 parts. These assume
an elongated form and enter the red cells of the host. The forms taken up by
Lamus multiply in the intestine and then pass to the salivary glands after about eight
days. The bug is then infectious when it bites. Brumpt notes that infection may
occur from inoculation of the faeces passed by the bug, especially through the
conjunctiva.

Trypanoplasma

The genus Trypanoplasma has a rather large blepharoplast, from which arise two
flagella. One extends forward as a free anterior flagellum, while the other projects
posteriorly, running along the border of the undulating membrane. This genus is
not known for man.

Leishmania

The parasites which cause a general infection in kala-azar and
leishmania infantile splenic anaemia but a local one in oriental sore
are usually separated as distinct species, Leishmania donovani for
kala-azar, L. infantum for infantile splenic anaemia and L. tropica
for oriental sore.

268 THE PROTOZOA

.

> x
^rt_

These parasites are grouped with the haemoflagellates and occur in their verte-
brate hosts exclusively as small, oval, cockle-shell-shaped bodies, measuring 2.5
3.0 microns. The protoplasm stains a faint blue and contains a rather large tropho-
nucleus which is peripherally placed and gives the appearance of the hinge of the
cockle shell. Besides this macronucleus we have a second chromatin staining body
which is often rod-shaped and set at a tangent to the larger nuclear structure. It is
called the blepharoplast or micronucleus and stains a more intense reddish than the
rather fainter stained pinkish macronucleus. One or more vacuoles are common in
the cytoplasm.

Some consider these nonflagellated bodies, which are usually found packed in
endothelial cells of spleen, liver, lymphatic glands and bone marrow, as resting stages,
the flagellate existence occurring in some other host than its vertebrate one. Patton
has carried on an immense amount of experimental work with the bedbug and has
noted the development of flagellate forms from the fifth to the eighth days in bugs
which fed on kala-azar patients showing leishman bodies in their peripheral circula-
tion. If the bugs are allowed a second feeding after the infecting blood meal the
flagellates disappear within twelve hours, so that for full development in the bedbug a
single feeding is requisite. He states that the flagellate forms change to post-flagel-
lates ones by the twelfth day. At the same time, although much evidence exists in
favor of the bedbug as host for the flagellate forms, it has not been shown experi-
mentally that the bedbug is definitely connected with the transmission of the disease.

Donovan is disposed to incriminate Conorrhinus rubrifasciatus as the transmitting
agent and furthermore he feels that there has not been sufficient investigation of mos-
quitoes along this line.

In the regions where leishmaniasis of infants occurs there is also
found a similar disease of dogs and Basil e has claimed that the disease
is transmitted from dog to dog by the dog flea.

As the dog has been regarded by some as the reservoir of the virus so naturally the
transmission of the disease from dog to child through the flea has been considered.
Wenyon, however, tried to infect two young dogs with great numbers of fleas which
had previously fed on dogs infected with canine leishmaniasis and at autopsy, five or
six weeks later, was unable to find parasites in smears from spleen, liver or bone
marrow and did not succeed in obtaining cultures from this material inoculated into
tubes of N. N. N. medium.

As regards oriental sore Wenyon has found that bedbugs and Stegomyia will feed
from the sores and take up parasites which develop into flagellate forms in the gut of
the insects.

Proof of transmission by these agents, however is lacking and others are inclined
to suspect the house fly or some species of moth midge.

In Brazil there exists some evidence that the cutaneous leishmaniasis found there
may be transmitted by species of the tabanid family.

It must be understood that there is always a suspicion that the
flagellate forms noted in arthropod experiments may be those of non-
pathogenic herpetomonad or crithidial species as such forms are

LEISHMANIASIS 269

common in arthropods and are difficult to distinguish from the flagellate
stage of leishman bodies.

The genera Herpelomonas (Leptomonas) and Crlthidia are frequently found in the
alimentary tract of insects and have caused confusion in the search for developmental
forms of various pathogenic flagellates in transmitting insects. In Herpetomonas, of
which the type species is H. muscoe domestic^, the body is spindle-shaped with a
rather blunt flagellar end and an attenuated anterior end. In Crithidia both extremi-
ties are pointed and the blepharoplast is situated toward the center quite near the
trophonucleus. In Herpetomonas the blepharoplast is near the rather blunt
flagellar extremity at some distance from the nucleus.

There is no undulating membrane in Herpetomonas and only a slightly developed
one in Crithidia while Trypanosoma has a fully developed one. In Herpetomonas
the blepharoplast is near the flagellated end and at a distance from the trophonucleus.
In Crithidia the blepharoplast is more posterior and near the macronucleus but still
anterior to it while in Trypanosoma the blepharoplast has moved so far posteriorly
as to pass the nucleus and be located at the posterior extremity. The flagellated
Leishmania is morphologically herpetomonad.

Very definite is our knowledge of the cultural forms of Leishmania. Rogers first
cultured material from splenic juice of kala-azar patients in 10% sodium citrate solu-
tion at a temperature of 1 7° to 24°C. The medium was slightly acidulated with citric
acid. There was no satisfactory development at blood temperature. In forty-eight
hours the oval parasites have developed into herpetomonad flagellates, from 20 to
22 microns long by 3 V£ microns broad, with a 2o-micron flagellum which takes origin
from the blunt anterior end of the body near the blepharoplast. The peripheral
blepharoplast and centrally placed macronucleus are at a distance from one another
as opposed to the approximation of the crithidial blepharoplast to the centrally
placed nucleus in a body with pointed anterior end.

Formerly it was thought that there were differences in the three
species of Leishmania from the standpoint of growth on various culture
media, L. donovani not growing on N. N. N. medium while L. infantum
grew well on N. N. N. medium but not in citrated blood. It is now
known that both species will grow on these two media.

It is absolutely essential in culturing L. donovani or L. infanttim that the blood
agar or citrated blood be sterile, as any bacterial contamination prevents growth.
With the parasite L. tropica, however, bacterial contamination does not inhibit
development and statements have even been made that growth is favored by a
staphylococcal symbiosis. L. tropica, it would seem, will develop into flagellated
forms in cultures at 28°C. while it will be remembered that Rogers in his original
experiments failed to obtain other than commencing signs of division at 27°C., 22°C.
being the temperature necessary for the development of flagellate forms.

L. tropica from South American cutaneous leishmaniases seems to grow more luxur-
iantly on N. N. N. medium than does that of oriental sore of Asia and Africa.

While differences in development on different culture media may
obtain not only with different species but with different strains of the

270 THE PROTOZOA

same species, it would appear that such variations cannot be utilized
as a means of separating the three species.

With animal inoculations we formerly thought that the parasite of kala-azar
could be differentiated from that of infantile leishmaniasis by the fact that dogs
could not be infected with L. donovani, while they were susceptible to infections
with L. infantum. Recently Donovan and Patton have successfully inoculated dogs
with kala-azar splenic material. Patton found the parasites in the liver, spleen and
lymphatic glands as well as bone marrow of the inoculated dogs. Consequently we
cannot separate the two visceral leishmaniases from a standpoint of susceptibility
of the dog. Monkeys are susceptible to both diseases.

As regards separating oriental sore from the visceral leishmaniases Gonder has
shown that white mice may be infected with both kala-azar and oriental sore, there
being produced in each case a general infection with the presence of parasites in
spleen and liver. A point of difference, however, is that the oriental sore mice
develop lesions on feet, tail and head which was not observed with the kala-azar
mice. There are some reasons for thinking that in human cutaneous leishmaniasis
a generalized infection may precede the local manifestations.

A very interesting point is that the dogs in India never show a
natural infection with L. donovani while in the regions where L. in-
fantum is responsible for human infections the natural infection of
dogs is not uncommon, indeed many think the dog the reservoir of
virus for both L. infantum and L. tropica. It has been suggested that
the dogs of India, where kala-azar prevails, may be immune.

As regards morphology it is usually stated that the parasites of the three species of
Leishmania are practically identical. In cultures it has been noted that the flagella
of L. tropica are longer and more twisted than those of L. infantum. Again it has
been observed that the parasites of the Oriental and South American skin lesions may
at times show a flattened or band-like trophonucleus instead of the constant round
or oval one of the visceral leishmaniases.

Escomel has reported the finding of flagellated Leishmania in the South American
sores.

Laboratory Diagnosis. — The leukemias can be easily differentiated
by the blood picture, an important matter because the spleen of
splenomyelogenous leukemia is very friable and the danger from splenic
puncture is far greater in this condition than in kala-azar. Banti's
disease with its leucopenia shows a rather similar blood picture and
can only be surely differentiated by the finding of leishman bodies in
kala-azar.

Malta fever, typhoid and the paratyphoids are best differentiated by blood cul-
tures or agglutination tests.

Until recently it was recommended that for diagnosis our best procedure was

INTESTINAL FLAGELLATES 271

to make a splenic puncture. Manson and others have pointed out the dangers
from splenic puncture in kala-azar and have rather preferred puncture of the liver,
although recognizing that the chances of obtaining parasites from a liver puncture,
are less than from a splenic one.

Statistics have been given where a mortality approximating i% has followed
spleen puncture. Bousfield, however, using an all glass syringe with a i^-inch
needle did not have a fatality in 1 20 spleen punctures.

For diagnosis the spleen or liver juice, rather than pure blood, is
smeared on a slide and stained by some Romanowsky method, pref-
erably that of Giemsa.

Cultures on N. N. N. medium can also be made.

One should always first examine a smear of the peripheral blood for parasites in
polymorphonuclear or large mononuclear leukocytes. The Sudan Commission
found leishman bodies in the peripheral blood of 13 out of 15 cases so examined, but
rarely did they find more than one parasite-containing leukocyte to a slide.

Quite recently Wenyon and others have noted the desirability of
culturing the peripheral blood in N. N. N. medium. Diagnosis may
be made in this way, provided one wait from two to three weeks
before reporting negatively as to the presence of flagellated Leishmania
in the cultures. As before stated, strict asepis and a room temperature
are requisite for flagellate development.

It has been noted that artificial pustulation might assist in diagnosis by giving
a multitude of polymorphonuclear leukocytes for examination for phagocytized
Leishmania.

Cochran has recently noted the advisability of excising a lymphatic gland and
making gland smears to examine for Leishmania. Others have reported success
with gland puncture as utilized in the glands of trypanosomiasis.

NOTE. — Darling has reported from Panama a protozoon somewhat
like Leishmania in which the cells .of lungs, liver, spleen, and lym-
phatic glands contained numerous parasites about 3 to 4^ in diameter,
slightly oval in outline, and containing a large and small chromatin
staining mass. He has given it the name Histoplasma capsulata.

INTESTINAL FLAGELLATES

These parasites of the intestinal tract are separated according to
the number of their flagella.

These flagella can easily be counted in a preparation mounted in Gram's iodine
solution. For this purpose I take a clean slide and make a vaseline line across it
about i inch from the end. A drop of the iodine solution is placed on the slide about

272 THE PROTOZOA

% inch from the vaselined line and a suitable portion of the faeces to be examii
is emulsified in it. The edge of a square cover-glass is then applied to the vaselined
line and allowed to drop on the preparation. By pressure suitable thicknesses of
fluid can be examined. There is an absence of current motion. Better, when access-
ible, is it to use the dark field illuminator as in this way the flagella are distinctly
brought out. The india ink method is also applicable. Staining of smears by
Giemsa's method, following fixation in methyl alcohol or 5% formalin solution is
more satisfactory for flagellates than for amoebae, which, as before stated, should
be fixed in moist smears and stained by haematoxylin. This method of mounting
in iodine solution, however, is the one I always use for encysted amoebae.

The intestinal flagellates are classified according to number of
flagella, absence or presence of an undulating membrane and of a
blepharoplast. Three of these flagellates are but rarely found in the
stools and seem to be of little importance. They are (i) Cercomonas,
which has a single nucleus with one free flagellum and a second one
which turns backward to be attached to the body and then projects
posteriorly as a second free flagellum, (2) Bodo, which has a single nuc-
leus, but two anteriorly projecting flagella and (3) Prowazekia which
has, besides the nucleus, a blepharoplast from which arise two flagella.
These flagellates can be cultivated on media used for the cultural
amoebae and it is thought by some that they at times show amoebae-
like stages.

There is an organism, supposed to belong to the moulds, which may be mistaken
for an encysted flagellate. It is called Blastocystis hominis and has a large central
vacuole with a refractile narrow rim which contains one or more nuclei. When
stained by Giemsa's stain the central part is very faintly stained while the rim is
deep blue.

Trichomonas intestinalis. — This is a very common parasite in diarrhceal stools
but as to its pathogenicity there is much doubt. It is pear-shaped and about 9 by
14 microns. There are three flagella projecting anteriorly with a fourth one bordering
an undulating membrane and projecting posteriorly. It has a cytostome near the
nucleus. There is also a T. vaginalis which is found in vaginal secretion of acid
reaction, disappearing when the reaction becomes alkaline as at the time of
menstruation. It is somewhat larger than the intestinal form and is not infre-
quently found in urine.

Tetramitus mesnili. — This flagellate differs from the preceding one in not having
an undulating membrane or fourth flagellum. The three anteriorly projecting
flagella are long and slender. There is a very prominent long slit-like cytostome
within which is a flagellum. The nonflagellate end is very much attenuated.

This parasite has been reported not infrequently as a cause of diarrhceal condi-
tions since its first reporting by Wenyon in 1910.

All the above-mentioned flagellates are found in the large intestines especially in
the region of the caecum.

CTLIATES

Lamblia

273

Lamblia intestinalis.— These parasites are about 10 X ISM and have a pear-shaped
body with a depression at the blunt anterior end. This depression enables the
flagellate to attach itself to the summit of an epithelial cell. Around the depression
are three pairs of flagella which are constantly in motion. Another pair of flagella
project from either side of the blunt little tail-like projection. When stained, the
parasites have a pyriform shape with two chromatin staining areas on either side
of the anterior end.

In motion they have a slow tumbling sort of progression. These parasites live
in the upper portion of the small intestines. The cysts are oval and show the
folded flagellate within. There is an appearance of two curved lines and two dots.
This infection is frequently associated with a debilitating diarrhoea. Some cases
show marked nervous symptoms. In examining the stools of 384 cases, who had
practically all been in Gallipoli or Egypt, Woodcock and Penfold, in the King George
Hospital, found 98 infected with protozoa as follows: Lamblia, 22; Trichomonas, 14;
Tetramitus (Macrostoma), n; E. coli, 57; E. histolytica, 8; Isospora, 10.

INFUSORIA (CILIATA)
The Infusoria are the most highly developed of the Protozoa.

The bodies of Infusoria are oval and may be free or attached to a stalk-like con-
tractile pedicle, as with Vorticella, or they may be sessile. The cilia, which are
characteristic, may be markedly developed around the cytostome (mouth) and serve
the purpose of directing food into the interior, while others act as locomotor organs.
The body is enveloped by a cuticle which may only have one opening or slit, to serve
as mouth; or it may have a second one, a cytopyge or anus. Usually the faecal
matter is ejected through a pore which may be visible only when in use. They usu-
ally have a large nucleus and a small one. Infusoria tend to encyst when conditions
are unfavorable (as when water dries up in a pond). When the cilia are evenly
distributed over the entire body of the cilia tes we have the order Holotricha; when
ciliated all over, but with more prominent cilia surrounding the peristome, we call
the order Heterotricha. It is to this order that the Infusoria of man belong.

Balantidium coli. — This is the only cilia te of importance in man.
It is a common parasite of hogs. It is from 60 to ioo/* long by 50 to
yo/* broad, and has a peristome at its anterior end which becomes narrow
as it passes backward. It has an anus. The ectosarc and the endosarc
are distinctly marked. The cuticle is longitudinally striated.

These parasites cause an affection similar to dysentery and may bring about a
fatal termination. It is almost impossible to escape noticing the actively moving
bodies if a fgecal examination is made. When encysted they are round.

Another ciliate, the B. minimum, 25 X i5M, has also been reported for man.

Nyctotherus faba has a kidney-shaped body and is about 25 by 15/1. It has a
large contractile vacuole at the posterior end. It has a large nucleus in the center
18

DESCRIPTION OF PLATE I

(Kolle and Wassermann)
Malarial Parasites

1. Two tertian parasites about thirty-six hours old, attacked blood-corpuscles
swollen.

2. Tertian parasite about thirty-six hours old; stained by Romanowsky's method.
The black granule in the parasite is not pigment but chromatin. Next to it and to
the left is a large lymphocyte, and under it the black spot is a blood plate.

3. Tertian parasite, division form nearby is a polynuclear leukocyte.

4. Quartan parasite, ribbon form.

5. Quartan parasite, undergoing division.

6. Tropical fever parasite. (^Estivo-autumnal.) In one blood-corpuscle may
be seen a smaller, medium, and large tropical fever-ring parasite.

7. Tropical fever parasite. Gametes half-moon spherical form. Smear from
bone marrow.

8. Tropical fever parasite which is preparing for division heaped up in the blood
capillaries of the brain.

Asexual Forms

9. Smaller tertian ring about twelve hours old.

10. Tertian parasite about thirty-six hours old, so-called amoeboid form.

11. Tertian parasite still showing ring form forty-two hours old.

12. Tertian parasite, two hours before febrile attack. The pigment is beginning
to arrange itself in streaks or lines.

13. Tertian parasite further advanced in division. Pigment collected in large
quantities.

14. Further advanced in the division. (Tertian parasite.)

274

PLATE I.

DESCRIPTION OF PLATE H

(Kolle and Wassermann)
Malarial Parasites

15. Complete division of the parasite. Typical mulberry form.

1 6. To the left is the completed division form, an almost developed gamete, which
is to be recognized by its dispersed pigment.

17. A tertian ring parasite, small size broken up.

1 8. Threefold infection with tertian parasite. The oval black granules are the
chromatin granules.

19. To the left, tertian parasite with large, sharply demarked, and deeply colored
chromatin granules. To the right, tertian parasite. Both thirty-six hours old.
Both probably gametes.

20. Tertian parasite thirty-six hours old, ring form.

21. Tertian parasite with beginning chromatin division, with eight chromatin
segments.

22. Tertian parasite chromatin division farther advanced with twelve chromatin
granules, in part triangular in form.

23. Completed division figure of a tertian parasite. Twenty-two chromatin
granules.

24. The young tertian parasites separating themselves from each other. The
pigment remains behind in the middle.

25. Quartan ring parasite, which is hard to differentiate from large tropical ring
or small tertian ring.

26. Quartan ring lengthening itself.

27. Small quartan ribbon form.

28. The quartan ribbon increases in width. The dark places consist almost
entirely of pigment.

276

PLATE II.

DESCRIPTION OF PLATE HI

(Kolle and Wassermann)
Malarial Parasite

29> 3°> 31- The quartan ribbon increases in width. The dark places consist
almost entirely of pigment.

32. Beginning division of the quartan parasite and the black spot in the middle
is the collected pigment.

33. Quartan ring.

34. Double infection with quartan parasites.

35. Wide quartan band. The fine black stippling in the upper half of the parasite
is pigment.

36. Beginning division of the quartan parasite. The chromatin (black fleck)
is split into 4 parts.

37. Division advanced, quartan parasites.

38. Typical division figure of the quartan parasite.

39. Finished division of the quartan parasite. Ten young parasites, pigment in
the middle.

40. Young parasites separated from one another.

41. Small and medium tropical ring, the latter in a transition stage to a large
tropical ring.

42. Small, medium and large tropical ring, together in one corpuscle.

278

PLATE III.

DESCRIPTION OF PLATE IV.

(Kolle and Wassermann.)
Malarial Parasite.

43. To the left a young tropical parasite. To the right a medium and large
tropical parasite.

44. An almost fully developed tropical parasite. The black granules are pig-
ment heaps.

45. Young parasites separated from one another. Broken up division forms
twenty-one new parasites.

46. To the left a red blood-corpuscle with basophilic, karyochromatophilic gran-
ules. Prototype of malarial parasite. On the right a red blood-corpuscle with re-
mains of nucleus.

Sexual Forms or Gametes.

47. An earlier quartan gamete (macrogamete in sphere form), female.

48. An earlier quartan gamete (microgametocyte), male.

49. Tertian gamete, male form (microgametocyte).

50. Tertian gamete, female (macrogamete).

51. Tertian gamete (microgametocyte) still within a red blood-corpuscle.

52. Macrogamete tertian within a red blood-corpuscle.

53. Tropical fever. (^Estivo-autumnal) gamete, half-moon (crescent) still
lying in a red blood-corpuscle. In the middle is the pigment. The concave side
of the crescent is spanned by the border of the red blood-corpuscle.

54. Gamete, tropical fever parasite.

55. Gamete of tropical fever parasite heavily pigmented.

56. Gamete of the tropical fever parasite (flagellated form), microgametocyte
sending out microgametes (flagella or spermatozoa).

280

PLATE IV.

282 THE PROTOZOA

with a small fusiform micronucleus lying close to it. It has only been reported
once for man.

SPOROZOA

This class of Protozoa gets its name from the method of reproduction
— sporulation. These parasites rarely show binary fission. While
the sporozoa are found within cells, in the tissues and in internal cavi-
ties, as intestine and bile ducts, yet it is as inhabitants of the blood that
they have their greatest importance for man — these are known as
Haemospbridia. A sporozoon may be either naked or amoeboid or be
covered with a distinct cuticle.

NOTE. — Sporozoa are divided into two subclasses — the Telosporidia and the Neo-
sporidia. In the former the vegetative activity of the protozoon goes on to full
growth at which time the reproductive activity commences. With the Neosporidia,
however, the growth and reproduction go on at the same time.

Among the Telosporidia we have the orders Gregarinaria, Coccidiaria, and
Haemosporidia.

Gregarines are chiefly parasites of arthropods and worms and are not known for
man or the higher vertebrates.

The subclass Neosporidia is practically of no importance in human parasitology,
only the order Sarcosporidia having been reported for man. From an economic
standpoint, however, the order Myxosporidia is of great importance — Nosema
bombycis being the cause of pebrine, a disease destructive to the silkworm.

Coccidiaria

The parasites of the order Coccidiaria are almost exclusively found in
the intestines and in the organs connected with it. In the vegetative
stage it lives within an epithelial cell, which it destroys. Afterward
it falls into the lumen lined by this epithelial cell and sporulates, either
by the method of schizogony or sporogony.

Owing to their egg-like shape, coccidia have often been considered as the ova of
intestinal parasites, and vice versa. Upon swallowing an oocyst with its contained
sporozoites the membrane of the oocyst is digested in the duodenum and the sporo-
zoites liberated. They enter epithelial cells, as of intestine, and reproduce by
schizogony. After a varying number of nonsexual cycles sporogony commences,
sporonts being produced instead of schizonts. The female sporont is fertilized by
the microgamete which is an elongated body provided with two flagella. These
microgametes are formed from the male sporont and when thrown off from the
periphery they enter (usually a single one) the macrogamete. After fertilization a
resistant membrane is formed and the term oocyst is used. Within the oocyst
are found smaller cysts, the sporocysts, in which the sporozoites are formed.

The cycle is very similar to that of malaria except that no arthropod host is

THE MALARIAL PARASITE 283

required for the sexual cycle. The spores which are formed in schizogony are known
as merozoites.

Merozoites may best be distinguished from sporozoites by the presence of a
nuclear karyosome, this being absent in sporozoites. In Eimeria we have the
oocyst containing four sporocysts with two sporozoites in each sporocyst while in
Isospora we have an oocyst containing two sporocysts with four sporozoites in each.

Eimeria stiedae. — This sporozoon is usually known as the Coccidium cuniculi
or C. omforme. It is most frequently found in the epithelium of the bile ducts.
It has very rarely been reported for man. In these cases (about five) cysts of the
liver have been found containing coccidia. The parasite is about 40 X 20/z, and is
oval in shape with a double outlined integument. The sporozoites, which form
inside, are falciform in shape. These escape and enter fresh epithelial cells, and
thus the process of schizogony goes on. The parasites of the liver are larger than
those found in the intestines, these latter being only about 30 X 15/1- In the fceces
the form most often found is the oocyst, about 40 X 2o/*. Infection takes place by
ingestion of the oocyst.

Wenyon states that the Eimeria oocysts, found in cases from Gallipoli, are round
instead of the usual oval. They are about 20 microns in diameter and contain four
sporocysts, 10X7 microns, each of which has two sporozoites with one or two highly
refractile residual bodies.

Isospora bigemina. — This parasite, formerly called the Coccidium bigeminum,
lives in the intestinal villi of dogs and cats. It is about 12X8/4 and shows a highly
refractile envelope (oocyst) containing two biscuit-shaped sporocysts within each of
which are four sporozoites. It has been reported for man three times.

Cases from the Gallipoli district also showed oocysts of Isospora with two sporocysts
and four sporozoites in each.

Haemosporidia

Of the Sporozoa found in the blood (Haemosporidia), the malarial
parasites are the only ones connected with disease in man.

There are at least three species of animal parasites which produce human malaria,
Plasmodium vivax, the cause of benign tertian, P. malaria of quartan and P. falci-
parum of sestivo-autumnal. These parasites belong to the haemamceba type of the
order Heemosporidia, of the class Sporozoa and of the phylum Protozoa.

This type of Haemosporidia is characterized by invasion of red
cells, amoeboid movement, pigment production and the extrusion of
flagellum-like processes from the male sporont after the blood is taken
from the animal and allowed to cool.

Other Hajmospordia which are very important in diseases of domesticated animals,
but not for man, are those of the piroplasm type.

These parasites of the red cells do not produce pigment and do not "exflagellate."
It is to parasites of this type that some authorities have ascribed the cause of black
water fever, a condition undoubtedly connected with malaria.

284 THE PROTOZOA

It has been thought proper by some to consider the malarial para-
sites as belonging to two genera, the genus Plasmodium, characterized
by round sexual forms and including P. vivax and P. malaria and the
genus Laverania, characterized by crescent-shaped sexual forms and
including but one species L. malaria, that of aestivo-autumnal malaria.

In addition to man, infections with parasites of a similar nature are found in mon-
keys (Plasmodium kochi; the sexual forms alone seem to be present), in birds
(Hcsmamaeba relicta; this organism is usually designated Proteosoma). An infection
of crows and pigeons of like nature is Halteridium. Numerous haemosporidia have
been reported for bats, various other mammals, tortoises, lizards, etc.

The life history of the malarial parasite is one of the most interesting chapters in
medicine. Laveran discovered the parasite in 1880. In 1885, Golgi noted that
sporulation occurred simultaneously at time of malarial paroxysm. Koch, Golgi,
and Celli demonstrated existence of different species for different types of fever.
King and Laveran (1884) considered possibility of mosquito transmission. Manson
(1894) formulated hypothesis that gametes were destined to undergo development in
the mosquito from observing that flagellated bodies only appeared some time after
the blood was withdrawn.

Ross (1895) demonstrated that flagellation takes place in the stomach of the mos-
quito. McCallum (1897) saw fertilization of macrogametes by microgametes of
Halteridium. Opie recognized differences in sexual characteristics.

Ross (1898) demonstrated life cycle of bird malaria (Proteosoma}, showing for-
mation of zygotes and presence of sporozoites in salivary glands. Grassi and Big-
nami proved the cycle for Anophelinas for human malaria. In 1900 (Sambon and
Low), infected mosquitoes from Italy were sent to London, where, by biting, they
infected two persons.

Life History. — Malaria can be transmitted by subcutaneous or
intravenous injection of the blood of a patient with the disease into a
well person, the same type being reproduced.

Such a method of transmission is only of scientific interest and the
regular method is as follows: An infected anopheline at the time of
feeding on the human blood introduces through a minute channel in the
hypopharynx the infecting sporozoite of the sexual cycle.

When man is first infected by sporozoites we have starting up a nonsexual cycle,
which is completed in from forty-eight to seventy-two hours, according to the species
of the parasite. The falciform sporozite bores into a red cell, assumes a round shape
and continues to enlarge (schizont). Approaching maturity, it shows division into a
varying number of spore-like bodies. At this stage the parasite is termed a mero-
cyte. When the merocyte ruptures, these spore-like bodies or merozoites enter a
fresh cell and develop as before.

Malarial Toxin. — At the time that the merocyte ruptures it is
supposed that a toxin is given off which causes the malarial paroxysm.

SEXUAL CYCLE OF MALARIAL PARASITE 285

Rosenau, by injecting intravenously filtered blood, taken from a patient at the
time of sporulation of the parasites caused a malarial paroxysm. No parasites
developed later. Another man who received a small amount of unfiltered blood
showed a slight paroxysm and four days later showed parasites in his blood. Hence
the parasite will not pass through the pores of a Berkefeld filter.

The cycle goes on by geometric progression from the first introduc-
tion of the sporozoite, but it is usually about two weeks before a
sufficient number of merocytes rupture simultaneously to produce
sufficient toxin for symptoms (period of incubation). This cycle is
termed schizogony. It is considered that there must be several
hundred parasites per cubic millimeter sporulating to be capable of
producing symptoms. ^

Gametes. — After a varying tfme, whether by reason of necessity for renewal of
vigor of the parasite by a respite from sporulation, or whether from a standpoint of
survival of the species, sexual forms (gametes) develop. Some think that sporozoites
of sexual and nonsexual character are injected at the same time. It is usually con-
sidered, however, that sexual forms develop from preexisting nonsexual parasites.
The developing gametes are often termed sporonts. Strictly, the sexual parasites
in the blood should be called gametocytes. The gametes take about twice as long to
reach maturity as schizonts. The life of a crescent has been estimated as about ten
days and that of the gametes of benign tertian and quartan about one-half this period.

The gametes show two types: the one which contains more pigment, has less
chromatin, and stains more deeply blue is the female — a macrogametocyte; the other
with more chromatin, less pigment, and staining grayish green or light blue is the
male — a microgametocyte. When the gametes are taken into the stomach of the
Anophelinse, the male cell throws off spermatozoa-like projections, which have an
active lashing movement and break off from the now useless cell carrier and are
thereafter termed microgametes. These fertilize the macrogametes and this body
now becomes a zygote. (Following nuclear reduction with formation of polar bodies
the macrogametocyte becomes a macrogamete.) This process of exflagellation can
be observed in a wet preparation under the microscope. There is first seen a very
active movement of the pigment of the male gamete and finally long delicate bulbous
tipped flagellum-like processes are thrown off (exflagellated) and push aside the red
cells by their progressive motion. McCallum saw a female Halteridium fertilized
by the microgamete, after which it was capable of a worm-like motion (vermiculus or
ookinete).

By a boring-like movement the vermiculus stage of the zygote goes through tt
walls of the mosquito's stomach, stopping just under the delicate outer layer of the
stomach or mid-gut. In three or four days after fertilization the zygote becomes
encapsulated and is then often called an oocyst. It continues to enlarge until at
about the end of one week it has grown to be about SOM in diameter and has be-
come packed with hundreds of delicate falciform bodies. Some only contain
a few hundred, others several thousand.

Zygotes.-- In some of his observations Darling has noted that the zygote of benign
tertian malaria grows larger and more rapidly than that of sestivo-autumnal and that

286

THE PROTOZOA

mn o 1

the pigment is clumped rather than in belts or lines as with aestivo-autumnal.
Darling has also noted that mosquitoes do not tend to become infected unless the
gamete carrying man has more than 1 2 gametes to the cubic millimeter.

The capsule of the mature zygote ruptures about the tenth day and the sporo-
zoites are thrown off into the body cavity. They make their way to the salivary
glands and thence, by way of the veneno-salivary duct, in the hypopharynx, they are
introduced into the circulation of the person bitten by the mosquito, and start a
nonsexual cycle. As the sexual life takes place in the mosquito, this insect in the
definitive host and man is only the intermediate host. The cycle in the mosquito
takes about ten to twelve days.

e

forms in Man
~ Gametes —

>/oheline mosquito (definitive
neriod tcLsts Q-/odays
t 25' 'to 30" C.

-sexua/ cyc/e 0 f
\jn man (inter- \ Q '
J^ mediary Aostjo0 o£

^— ,.

®-cg

FIG. 63. — Sexual (sporogony in mosquito) and non-sexual (schizogony in man)
cycle of the malarial parasite. The sporogony diagram to the left shows in lower
portion the fertilization of the female gamete by the microgamete. The vermiculus
stage of the zygote is shown boring into the walls of the mosquito's stomach to later
become the more mature zygote packed with sporozoites as shown in the upper dia-
gram of the developmental processes in the mosquito's stomach.

Efficient Mosquito Hosts. — It must be remembered that only certain
genera and species of Anophelinae are known malaria transmitters;
thus Stephens and Christophers, in dissecting 496 mosquitoes of the
species M. rossi, did not find a single gland infected with sporozoites.
With M. culicifacies, however, 12 in 259 showed infection. A
mosquito which is capable of carrying out the complete sporogonous
cycle is an efficient host and in the case of malaria the mosquito is the
definitive host (sexual life of parasite).

Malarial Index. — Mosquito dissection is one method of determining
the endemicity of malaria or the malarial index. There are two other
methods: i. by noting the prevalence of enlarged spleens, and 2. by

MALARIAL INDEX 287

determining the number of inhabitants showing malarial parasites in
the blood. This index is best determined from children between two
and ten years of age, as children under two show too high a proportion
of parasites in the peripheral blood while those over ten years of age
show too great an incidence of enlarged spleens.

As before stated there are three species of malarial parasites: i. Plasmodium
vivax, that of benign tertian — cycle, forty-eight hours; 2. Plasmodium malaria, that
of quartan — cycle, seventy-two hours; and 3. Plasmodium f aid par um, that of aestivo-
autumnal or malignant tertian — cycle of forty-eight hours.

Multiple Infections. — Variations in cycles may be produced by infected mosquitoes
biting on successive nights,J|p that one crop will mature and sporulate twenty-four
hours before the second. Tr^is would give a quotidian type of fever. In aestivo-
autumnal infections anticipation and retardation in the sporulation cause a very
protracted paroxysm, lasting eighteen to thirty-six hours; this tends to give a con-
tinued or remittent fever instead of the characteristic intermittent type.

Plasmodium Vivax. — In fresh, unstained preparations, taken at the time of the
paroxysm or shortly afterward, the benign tertian schizont, or nonsexual parasite,
is seen as a grayish white, round or oval body, whose outlines cannot be distinctly
differentiated from the infected red cell. They are about one-fifth of the diameter
of the red cell and are best picked up by noting their amoeboid activity. In about
eighteen hours fine pigment particles appear and make them more distinct. After
twenty-four hours the lively motion of the pigment and the projection of pseudopod-
like processes, in a pale and swollen red cell, makes their recognition very easy.
When about thirty to thirty ix hours old the amoeboid movement ceases.
Approaching the merocyte stage the pigment tends to clump into one or two pigment
masses and one can recognize small, oval, highly refractile bodies within the sporulat-
ing parasite.

The gametes or sexual forms do not show amoeboid movement, but the fully
developed gamete, which is generally larger than the red cells, has abundant pig-
ment, which is actively motile in the male gamete and nonmotile in the female.
The male gamete is more refractile, is rarely larger than a red cell and shows yellow
brown, short rod-like particles of pigment. Abou^f teen minutes after the making
of a fresh preparation these male gametes "throw out four to eight long, slender,
lashing processes, which are about 15 to 20 microns long. These spermatozoon-like
bodies now break off from the useless parent cell and with a serpent-like motion glide
away in search of a female gamete, knocking the red cells about in their passage
through the blood plasma.

The female gamete is larger than a red cell, is rather granular and has more abi
dant dark brown pigment than the male.

Stained Smears.— In dried smears, stained by some Romanowsky method, as that
of Wright, Leishman or Giemsa, we note small oval blue rings, about one-fifth of the
diameter of the infected yellowish-pink erythrocyte. One side of the ring is dis
tinctly broader than the rather fine opposite end, which seems to hold a round,
yellowish-brown dot, the chromatin dot, and has a resemblance to a signet ring.
These small tertian rings of the nonsexual parasites (schizont) are seen about the

288 THE PROTOZOA

vtotin

time of the commencement of the sweating stage of the paroxysm. Two chromatin
dots in the line of the ring are rare as is also true of more than one ring in a red cell.

When the parasite is about twenty-four hours old we note that it contains much
pigment and has an amoeboid or multiple figure of eight contour, is about three-
fourths the size of a red cell and that the infected red cell is about one and one-half
times as large as in the beginning and presents a washed-out appearance. It is an
anaemic-looking cell. We also note, as characteristic of a benign tertian infection,
reddish-yellow dots in the pale red cell, which are known as Schiiffner's dots.
These, practically, are characteristic for benign tertian.

A few hours before the completion of its forty-eight-hour cycle the contained
pigment begins to clump, the chromatin to divide and, finally, we have a sporulating
parasite, in which the 16 to 20 small, round, bluish bodies, with chromatin dots, are
irregularly distributed over the area of the merocyte.

The gametes, or sexual parasites, show a thicker blue ring and have the chromatin
dot in the center of the ring. The pigmentation of the half-grown gametes is more
marked than that of schizonts of equal size. The shape of the gametes is not
amoeboid, as is that of the twenty-four- to thirty-six-hour-old schizont, but round
or oval. The full-grown gametes have the pigment distributed and the chromatin
in a single aggregation — -just the opposite of nonsexual parasites. The male gamete
stains a light grayish blue and has a very large amount of chromatin, usually cen-
trally placed. The female gamete stains a pure blue, has only about one-tenth as
much chromatin as plasma, with the chromatin often placed at one side. The pig-
ment of the female gamete is dark brown while that of the male is yellowish brown.

Plasmodium Malariae. — In fresh preparations the young quartan schizont has
only slight amoeboid movement and, as development proceeds, the rather dark
brown, coarse pigment tends to arrange itself peripherally about the band-shaped
or oval parasite.

The infected red cell shows but little change. At the end of seventy-two hours
the rather regular daisy form of the merocyte is more distinct than that of the benign
tertian merocyte.

The distinctions between the male and female gametes are similar to those of the
benign tertian gametes. In Romanowsky stained smears it is difficult to distinguish
the young quartan schizont from the benign tertian one but, after twenty-four
hours, the tendency of the quartan schizont to assume equatorial band forms across
a red cell of normal size and staining characteristics and without Schiiffner's dots
makes the differentiation easy. In the fully developed sporulating parasite or
merocyte the eight merozoites assume a regular distribution giving it a daisy
appearance.

The gametes show practically the characteristics of the benign tertian ones but
are smaller.

Plasmodium Falciparum.— The young schizont of malignant tertian is extremely
difficult to detect in fresh preparations, there being noted early in the rather long
continued, hot stage, only small crater-like dots, about one-sixth of the diameter of
a red cell which, however, show an active amoeboid movement.

Later on in the hot stage these ring-like dots enlarge to become about one-third
of the diameter of a red cell, most often occupying the periphery of the infected
red cell. About this time, or at the very commencement of the pigmentation, the

CULTIVATION OF MALARIAL PARASITE 289

schizont containing red cells disappear from the peripheral circulation so that the
further development is rarely observed in blood specimens.

The infected cell is brassy in color and shrunken in shape — it shows evidences
of degeneration. The gametes appear as crescent-shaped bodies, which are abso-
lutely characteristic of malignant tertian, the male gamete being more hyaline and
delicate while the female one is more granular and larger.

In Romanowsky stained preparations we see, while the fever is sustained, small
hair-like rings, with geometrical outline, with frequently two chromatin dots in one
end of the ring and a single red cell often showing two or more of these young rings.
The rings are often seen as if plastered on the periphery of the red cells or as if having
destroyed a rounded section of the rim of the red cell. As the fever declines the
rings tend to disappear from the peripheral circulation. The infected red cells often
show polychromatophilia and distortion.

In old aestivo-autumnal cases, or those with severe infection, we may see adult
rings and merocytes, which latter are smaller than those of benign tertian, show from
1 6 to 32 irregularly placed merozoits and a sharply clumped mass of pigment.

The gametes are the striking crescent-shaped bodies and these show the distinc-
tions of blue staining for the female, with lighter gray-green staining and abundance
of chromatin for the male. The chromatin staining of crescents does not stand out
so well as that of the round form gametes of benign tertian and quartan.

As regards differentiation of species and cycle the examination of
stained smears is more satif actory and definite, as well as less time con-
suming. Still, one obtains many points of differentiation in the fresh
preparation and should study such a preparation while carrying out the
staining of his dried smear.

Central vacuolation of red cells is common in malarial anaemia and may be mis-
taken for nonpigmented parasites.

Malarial rings are usually peripheral and do not vary in size as one focuses up and
down as do the central vacuoles.

A very puzzling but well-recognized finding in cases treated with quinine or sal-
varsan is the so-called quinine affected parasite. Such parasites lack definiteness
of outline and show poor chromatin staining. The gametes do not seem to show
these effects from the drug.

Cultivation. — As to cultivation of malarial parasites. Bass takes from 10 to 20
c.c. of blood from the malarial patient's vein in a centrifuge tube which contains
Ho c.c. of 50% glucose solution. A glass rod, or a piece of tubing extending to
the bottom of the centrifuge tube is used to defibrinate the blood. After centri-
fugalizing there should be at least i inch of serum above the cell sediment. The
parasites develop in the upper cell layer, about >£0 to Ko inch from the top. All of
the parasites contained in the deeper lying red cells die. To observe the develop-
ment, red cells from this upper Ko-inch portion are drawn up with a capillary bulb
pipette.

Should the cultivation of more than one generation be desired, the leukocyte uppe
layer must be carefully pipetted off, as the leukocytes immediately destroy the
merozoites. Only the parasites within red cells escape phagocytosis.

290

THE PROTOZOA

parasites are much more resistant. Bass thinks he observed parthenogen
The temperature should be from 40° to 4i°C. and strict anaerobic conditions ob-
served. ^Estivo-autumnal organisms are more resistant than benign tertian ones.
Dextrose seems to be an essential for the development of the parasites.

Bass considers that P. vivax has a disc-like structure which enables it to squeeze
through the brain capillaries while adult schizonts of P. falciparum have a solid
oval form which causes them to be caught in the capillaries.

The Thompsons have rather .simplified the method of Bass. They draw 10 c.c.
of blood into a test-tube containing the usual amount of glucose solution. They
then defibrinate the blood by stirring with a thick wire for about five minutes and
remove the wire with the adhering clot. They then pour this defibrinated blood
into several small sterile test-tubes, which should contain at least a i-inch column.
Rubber caps are adjusted over the cotton plugs and the tubes placed in the incu-
bator. They note the tendency of cultures of P. falciparum to agglutinate which
is not true of P. vivax.

They think this agglutination the great cause of the plugging of capillaries in
pernicious malaria. They note 32 merozoites as maximum number in sporulation
of P. falciparum while P. vivax has usually 16 or more, but never as many as 32

This would explain the shorter incubation period of malignant tertian. The
pigment of P. falciparum clumps much earlier in. the developing schizont than that
of P. vivax and is much coarser and more discrete.

While Bass thought he noted parthenogenesis in cultures others have failed to
observe any evidence of it.

UNSTAINED SPECIMEN (FRESH BLOOD)

P. vivax
(benign tertian)

P. malariae
(quartan)

P. falciparum
(malignant tertian)
(aestivo-autumnal)

Character of the
infected red cell.

Swollen and light in
color after eighteen
hours.

About the size and
color of a normal red
cell.

Tendency to distortion of
red cell rather than crena-
tion. Shriveled appear-
ance. (Brassy color.)

Character of young
schizont.

Indistinct amoeboid
outline. Hyaline.
Rarely more than
one in r.c. Active
amoeboid move-
ment. One-third
diam. of r.c.

Distinct frosted glass
disc. Very slight
amoeboid motion.

Small, distinctly round,
crater-like dots not more
than one-sixth diameter
of red cell. Two to four
parasites in one red cell
common. Shows amoe-
boid movement until ap-
pearance of pigment.

Character of ma-
ture schizont.

Amoeboid outline.
No amoeboid move-
ment.

Rather oval in shape.
Sluggish movement
of peripherally
placed coarse black
pigment.

Only seen in overwhelming
infections. Have scanty
fine black pigment
clumped together.

Pigment.

Fine yellow-brown,
rod-like granules
which show active
motion in one-half-
grown schizont.
Motion ceases in
full-grown schizont.

Coarse almost black
granules. Shows
movement only in
young to half-grown
schizont.

Pigmented schizonts very
rare in peripheral circula-
tion except in overwhelm-
ing infections. Tends to
clump as eccentric pig-
ment masses almost black
in color.

PIROPLASMS
STAINED SPECIMEN

291

P. vivax
(benign tertian)

P. malarias
(quartan)

P. falciparum
(malignant tertian)
(aestivo-autumnal)

Character of in-
fected red cell.

Larger and lighter
pink than normal
red cell. Shows
"Schuffner's dots."

About normal size
and staining.

Shows distortion and some
polychromatophilia and
stippling. Rarely we
have coarse cleft -like red-
dish dots— Maurer's spots.

Character of young
schizont.

Chromatin mass usu-
ally single and situ-
ated in line with the
ring of the irregularly
outlined blue para-
site.

Rather thick round
rings which soon
tend to show as
equatorial bands.

Very small sharp hair-like
rings, with a chromatin
mass protruding from the
ring. Often appears on
periphery of red cell as a
curved blue line with
prominent chromatin dot.
Frequently two chro-
matin dots.

Character of half-
grown schizont.

Vacuolated or Fig. 8
l9op-like body with
single chromatin ag-
gregation. Schuff-
ner's dots.

More marked band
forms stretching ac-
ross r.b.c.

Not often found in periph-
eral circulation. C h r o -
matin still compact.

Character of ma-
ture schizont.

Fine pigment rather
evenly distributed
in irregularly out-
lined parasite.

Coarse pigment
rather peripherally
arranged in an oval
parasite.

Very rarely seen in periph-
eral circulation in ordi-
nary infection. Pigment
clumps early.

Character of mero-
cyte.

Irregular division into
15 or more spore-
like chromatin dot
segments.
Mulberry.

Rather regular di-
vision into eight or
10 merozoites —
Daisy.

Sporulation occurs in
spleen, brain, etc. Rarely
in peripheral circulation.
Eight to 10 chromatin
staining merozoites.
(In culture 32.)

Character of mac-
rogamete.

Round deep blue.
Abundant, rather
coarse pigment,
chromatin at per-
iphery.

Round, similar to P.
vivax but smaller.

Crescentic, deep blue, pig-
ment clumped at center,
chromatin scanty and in
center.

Character of mi-
crogametocyte:

Round, light green-
blue, pigment less
abundant, c h r o -
matin abundant and
located centrally or
in a band.

Round like P. vivax.

More sausage-shaped than
crescent. Light blue.
Pigment scattered
throughout. Chromatin
scattered and in greater
quantity but difficult to
stain.

PIROPLASMS

Belonging like the malarial parasite to the Haemosporidia we have a
group of parasites known as the PIROPLASMS. The correct name for
these parasites is Babesia but they are better known under the name
Piroplasma. They are minute organisms, usually pear or rod shape,
which invade the red corpuscles. They produce no pigment but
destroy the corpuscle and set free the Hb. which is excreted in enor-
mous amounts by the kidneys. It is this which gives the name red-
water to the disease usually designated Texas fever of cattle.

2Q2

THE PROTOZOA

The cause of this disease is B. bovis (B. bigemina) and the parasite is transmit
by a tick, Mar gar opus annulatus. There is also a disease of dogs called malignant
jaundice of dogs which is caused by B. canis and also transmitted by a tick. Organ-
isms of this kind have been thought of in connection with blackwater fever of man.
Seidelin has claimed that a parasite of similar nature, Paraplasma flavigenum, was
the cause of yellow fever.

At one time spotted fever of the Rocky Mountains was supposed to be due to a
parasite named Babesia hominis. The parasite of Oroya fever is
probably protozoal in nature. It is found in red cells as rod-
like bodies and is called Bartonella bacllliformis . See Oroya
fever.

SARCOSPORIDIA

Sarcosporidia are sporozoa found in the striped
muscles of various mammals and birds. They are
common in the pig and mouse and have been re-
ported for man in three well-authenticated cases. In
the last, Darling found these protozoa in the biceps
muscle of a negro patient in Panama. In Baraban's
case the laryngeal muscles at autopsy were found
to show cysts about Jf 5 inch long which contained
sickle-shape sporozoites about qn long.

FIG. 63. — Mie- They are known also as Miescher's tubes when in muscle fibers,
scher's sac from They are divided into three genera: Miescheria and Sarcocystis
olTa 'ho501*1 XU1Q when Parasitic "* muscle fiberJ Balbiania, when parasitic in the
diameters. (After intervening connective tissue of the muscles. The method of
Ostertag.) transmission is unknown. In some places more than 50% of

the sheep and pigs may show infection.

Miescheria has a thin membrane surrounding the cyst while that of Sarcocyslis
is thickened and radially striated by small canaliculi.

As the young trophozoite grows nuclei increase and a definite membrane forms
which the sporoblasts eventually fill. According to Minchin the Sarcosporidia con-
tain only one genus, Sarcocystis. It is never parasitic for invertebrate hosts and
while occasionally found in birds and reptiles it is preeminently a parasite of the
higher vertebrates. As a rule, they are harmless parasites but the Sarcocystis muris
is very pathogenic for the mouse. Closely related to the order Sarcosporidia is the
parasite Rhinos poridium kinealyi.

Rhinosporidium kinealyi. — It causes pedunculated tumors of nasal cavity. The
pansporoblasts enlarge in the center of the connective tissue of the nasal polyp and
contain about 12 sporoblasts. When mature the cystic-like polyp bursts and the
sporoblasts are liberated to extend the infection.

CELL INCLUSION DISEASES 293

CHLAMYDOZOA

These organisms are generally considered as being protozoal in
nature and as a rule belong to the filterable viruses, which is the desig-
nation for the infectious principles of those diseases, in which filtration
of defibrinated blood or serum through a Berkefeld filter capable of
holding back so small an organism as the M. melitensis, does not pre-
vent the infection being transmitted when introduced by the proper
atrium of infection. The Chlamydozoa are also characterized by the
occurrence of "cell inclusions."

The best known infections of this group of diseases in man are smallpox, vaccinia,
rabies, trachoma, molluscum contagiosum, and foot and mouth disease. There are
many such infections in other animals. The cell inclusions are regarded as prod-
ucts of cellular reaction to a virus which is more or less impossible of demonstra-
tion. The discovery of exceedingly minute granules in some of these diseases, as
in variola and trachoma, has suggested that, as a reaction to the invasion by such
a granule, the cell throws an eveloping mantle about the invading particle. To
designate this we use the name Chlamydozoa.

The generic name Cytorrhyctes has been applied to certain of these viruses, thus
C. vaccinia develops within the epithelial cells of stratified epithelium. In vaccinia,
Councilman and his colleagues consider that the development only takes place in
the cytoplasm of the cell. In variola, however, the developmental cycle affects the
nucleus.

Cytorrhyctes luis, reported as the cause of syphilis, sporulates in the blood-vessels
and in the connective tissue, not in epithelial cells.

Cytorrhyctes scarlatina was reported by Mallory to have been found in the skin
in four cases of scarlet fever.

CHAPTER XVII

FLAT WORMS

CLASSIFICATION OF THE PLATYHELMINTHES (FLAT WORMS)

Class

Family

Trematoda

Cestoda

Fasciolidae

Paramphistomidae

Schistosomidae

Dibothriocephalidae

Tamiidae

Genus

Species

Fasciola
Fascioletta

F. hepatica
F. ilocana

Fasciolopsis
Dicroccelium

F. buski
D. lanceatum

Paragonimus
\ Opisthorchis

Clonorchis

Heterophyes
Cladorchis

P. westermanii
O. felineus
(C. sinensis
C. endemicus
H. heterophyes
C. watsoni

Gastrodiscus

G. hominis

{S. haematobium

Schistosoma

S. japonicum
S. mansoni

f Dibothriocephalus
\ Diplogonoporus
Dipylidium -

Hymenolepis

D. latus
D. grandis
D. caninum
H. nana
H. diminuta

Taenia
Davainea

T. solium
T. saginata
D. madagascariensis

NOTE. — Two larval Taeniidae are found in man (Cysticercus celluloses and Echino-

coccus polymorphous).

Also two larval Dibothriocephalidae (Sparganum mansoni and Sparganum prolifer.)
Two parasites often referred to as ophthalmic flukes have been reported lying

between the crystalline lens and its membrane. They have been considered as

possibly trematode larvae. Distomum ophlhalmobium was found in 1850 in the eye

of a child and Monostoma lentis in the eye of an old woman.

TREMATODES OR FLUKES

Flukes are generally leaf-like in outline, rarely cylindrical, and ex-
hibit marked variation in size and shape. They are nonsegmented and

294

FLUKES

295

do not have cilia on ectoderm. Very characteristic of them is the
possession of suckers by which they hold on to the skin or alimentary
system of their host.

3*3

They are divided into two orders: i. the Monogenea in which the egg gives rise
to a larva which later becomes the adult and 2. the Digenea. It is to this latter that
the flukes parasitic in man belong. This order is characterized by the fact that the
larva becomes parasitic in some second animal and then gives rise to a second gen-
eration of larvae which latter develop into adults.

296 FLAT WORMS

The largest human fluke, Faciolopsis buskl, is from 2 to 3 inches (50 to
75 mm.) in length, while the Heterophyes heterophyes is less than ^2 inch (2
mm.) in length. The most important fluke, the liver fluke, Clonorchis endemicus,
is flat and almost transparent, while the almost equally important lung fluke, the
Paragonimus westermanii, is oval, almost round and reddish brown in color. With
the exception of the Schistosomidae, all flukes are hermaphrodites, and, with the
exception of this family, all flukes have operculated eggs. The only other opercu-
lated (with a lid) eggs we meet with in man are those of the Dibothriocephalidse.

The three important families of flukes parasitic for man are:

1. Paramphistomidae — flukes with two suckers situated at either
extremity.

2. Fasciolidae — flukes with two suckers, one terminal, the other ad-
jacent to it and situated ventrally. This family includes the im-
portant genera Fasciola, Opisthorchis, Dicroccelium, Fasciolopsis, and
Paragonimm. In Paragonimus and Heterophyes the genital pore is
posterior to the acetabulum, in the other genera it is anterior. Fas-
ciola has a dendritic intestinal canal which is not the case with Clon-
orchis, Fasciolopsis, Fascioletta, Opisthorchis and Dicroccelium.
In Dicrocxlium the testicles are anterior to the uterus, in Opisthorchis,
Clonorchis, Fasciolopsis and Fasciohtta they are posterior. Fascio-
lopsis and Clonorchis have branched testicles (the former a very large
ftuke-Clonorchis of medium size) while those of Opisthorchis are lobed.

3. Schistosomidae: In this family we have a leaf -like male which by
a folding in of its sides makes a channel for the thread-like female. The
sexes are separate, not hermaphroditic as with the Fasciolidae and
Paramaphistomidae.

Flukes have two suckers which, except in the Paramphistomidae, are quite near
each other — one is termed the oral sucker and the other the ventral sucker or acet-
abulum. The intestinal tract consists of a pharynx, proceeding from the oral sucker
which bifurcates and terminates in blind intestinal caeca.

At the posterior extremity is an excretory pore which is at the termination of
a duct which divides into ramifying branches. This is the water-vascular system.
The testes, of various shapes and relations to the uterus, are more or less centrally
situated and have vasa deferentia. In some flukes the receptaculum seminis is a
conspicuous organ. The vitellaria are bilateral branching glands which pour
nutrient material into the ootype. It is in the ootype that the eggs are formed,
and opening into it we have the adjacent ovary. The shell gland is near the ovary.

A canal, known as Laurer's canal, leads from the ootype to the exterior, the
function of which is in question. It is probable that as trematodes have no sperma-
theca, the spermatozoa from other flukes enter by way of this canal. The life his-
tdry of the important human flukes is unknown. It is supposed that this, in a meas-
ure, may resemble that of the common liver fluke of sheep (sheep rot). In this the
eggs containing a ciliated embryo (miracidium) pass out in the faeces. This embryo

LIVER FLUKES 2Q7

is hatched out and, gaining the water, swims about actively until it reaches some
suitable mollusk (Limnaa truncatula) . By means of a pointed end, it bores its
way into the body of the gastropod and in the pulmonary chamber becomes a
bag-like structure (the sporocyst) from the germinal cells of which develop a creature
with an alimentary canal (redia). The rediae tend to break out of the sporocyst and
wander to the liver of the snail. These rediae may give rise to a second generation
of rediae.

From the rediae minute little worms resembling adult flukes in possessing suckers,
but differing in the possession of a tail, develop (cercaria).. Having reached maturity,
these cercariae leave the rediae, and, as in case of Faseiola hepatica, lose the tail, be-
come encysted on blades of grass, to be eaten by sheep and again commence the cycle.
The encysted cercariae develop into adult liver flukes. It is probable that with many
flukes the cercariae enter some host, as mollusk, insect, or fish, and that it is by eat-
ing such animals as food that man becomes infected. Looss thinks it possible that
the miracidium of Schistosoma hcematobium may bore its way directly into man,
as do the larvae of the hookworm. Manson also suggests that the reporting by Mus-
grave of 100 mature lung flukes in a psoas abscess makes it very probable that these
parasites entered the body as miracidia. The idea in China is that the infection
with the common liver fluke of man is brought about by eating fish. Fluke disease
is generally known as distomatosis or distomiasis.

LIVER FLUKES

Faseiola hepatica (Dislomum hepaticum). — This fluke, while of
enormous economic importance by reason of destruction of sheep, has
only been reported 23 times in man, and in these instances does not
seem to have occasioned marked symptoms.

It has a cone-shaped anterior projection and is about 1% inches (30 mm.) long.
The intestinal canal, as well as the testicles, is branched. These intestinal diver-
ticula are well marked in the cone just after the branching from the oesophagus.
Diameter of acetabulum about 1.6 mm., of oral sucker i mm. There is a possible
importance of F. hepatica in connection with a peculiar affection known as
"halzoun." This results from the eating of raw goat-liver, and it is supposed that
the flukes crawl up from the stomach and, entering the larynx or attaching themselves
about the glottis, produce the asphyxia characteristic of the disease. ^

Dicrocoslium lanceatum.— This has only been reported seven times in man.
The symptoms are unimportant. The fluke is about Yz inch (8 mm.) long, with
testicles anterior to the uterus.

Clonorchis endemicus (Opisthorchis sinensis).— This fluke and the
C. sinensis are the most important of the human liver flukes. Until
recently these flukes were known as Opisthorchis sinensis.

Looss has separated this genus from Opisthorchis principally by the character-
istic of branching testicles— those of Opisthorchis being lobed. This fluke is very
common in China and Japan— in certain sections of Japan 20% of the population

298 FLAT WORMS

being infected. This fluke is about y± to }£ inch (8 mm.) long and C. sinensis about
% inch long and K inch broad (16 X 4 mm.). When squeezed out of the
thickened bile ducts it is so transparent and glairy as almost to resemble glairy
mucus. As many as 4000 of these parasites have been found in a case, chiefly in
the liver, but at times in the pancreas. This fluke is supposed to produce most
serious symptoms, as indigestion, swelling and tenderness of liver, ascites, oedema,
and a fatal cachexia. As a matter of fact, many physicians in China attribute very
little pathogenic importance to it. The disease is diagnosed by the presence of the
ova in the stools. The source of infection is probably through the eating of uncooked
fish.

Kobayashi has examined various mollusks and fish for trematode larvae. He
succeeded in infecting nine kittens and two cats by feeding them with certain fresh-
water fishes whose flesh contained trematode larvae. These fish were found in
districts where human distomiasis was common. The view is taken that the two
species of Clonorchis are identical.

Opisthorchis felineus. — This fluke is smaller than the C. endemicns, and is a
common parasite of the gall-bladder and bile ducts of cats. There are two-lobed
testicles in this species instead of dendritic ones as in C. endemicus. In certain
parts of Siberia the parasite is found in more than 6% of the human autopsies. The
symptoms are similar to those caused by C. endemicus.

Other liver flukes of less importance which have been reported for man are: i.
Opisthorchis noverca. This was found in bile ducts of two natives of Calcutta. It
was lancet-shaped and covered with spines.

2. Metorchis truncatus: This is a small fluke, K2 incn (2 mm.) long, squarely
cut across at Its posterior end and covered with spines. This was possibly found
once in man.

Intestinal Flukes

Cladorchis watsoni (Amphistomum watsoni). — This fluke is about ^ inch
(8 mm.) long, of oval outline but broader at posterior end and has an indistinct oral
sucker and a large sucker at the other end. This parasite has been reported from
northern Nigeria and is said to be a common infection of regions about Lake Chad.
Eggs, 125 X 75M-

Gastrodiscus hominis (Amphistomum hominis). — This fluke is about ^ inch
(6 mm.) long and has a disc-like concavity, about Y§ inch in diameter from
which proceeds a teat-like projection, bearing an oral sucker. The acetabulum
is in the posterior border of the disc. While it has only been reported a few times for
man, indications are that it is probably fairly common in India and Assam.
Eggs, 150 X 72/x. It gives rise to dysenteric symptoms.

Fasciolopsis buski (Distomum crassum). — This is probably a rather common
parasite in India, as Dobson found the eggs in i% of the stools of more than
1000 coolies. The fluke is from 2 to 3 inches (40 to 70 mm.) in length and about ^
inch (12 mm.) in breadth. It is thick, brown in color, and has a very large acetabu-
lum, three times the size of the oral sucker and located almost adjacent to it. The
branched ovary and shell gland lie in the center with the branched testicles posterior.
The coiled uterus is anterior to the testicles. Eggs, 125 X 75M- These parasites
cause dyspeptic symptoms and an irregular diarrhoea. It is also called Distomum

LUNG FLUKES 299

crassum. F. rathouisi is now considered to have been a shrunken F. buski, as it
seems to be anatomically similar to F. buski, Kwan's fluke reported from Hong
Kong, was possibly F. buski.

Heterophyes heterophyes (Cotylogonimus heterophyes).— This exceedingly
small fluke (2 X 0.5 mm.), which can be recognized by its small size (less than ^2
inch long) and large, prominent acetabulum, was formerly supposed to be rare.
The oral sucker is much smaller than the acetabulum. The elliptical testicles lie
at the extreme posterior end. Cuticle has scale-like spines. The eggs are 30 X I7M-
Very characteristic of this genus is the large sucker-like genital pore just below and
to one side of the acetabulum. Looss has shown that it is quite common in Egypt,
he having found it twice in Alexandria in nine autopsies. The parasites occupy the
ileum. It is common in dogs.

Echinostoma ilocana (Fascioletta ilocana).— This is a small fluke, about Y± inch
(6 mm.) long. There are two massive testicles in the posterior part of body. The
acetabulum is prominent and about 5ooju in diameter. This fluke has a ring of
spines around the anterior extremity. Ovary anterior to testes. Genital pore
anterior to acetabulum. The egg of this small fluke is quite large (ioo/i) and has an
operculum. These trematodes were found by Garrison in five natives of Luzon,
P. I., after treatment with male fern.

LUNG FLUKES

Paragonimus westermanii (Distoma ringeri). — In certain parts of
Japan and Formosa it is estimated that as many as 10% of the inhab-
itants may harbor this parasite.

It is also common in China, and recently many cases have been reported in the
Philippines. Dr. Stiles states that around Cincinnati, Ohio, there was at one time
quite a heavy infection among the hogs, so that it may be that certain cases diagnosed
in man as pulmonary tuberculosis are paragonimiasis.

It is popularly known as endemic haemoptysis on account of the accompanying
symptoms of chronic cough and expectoration of a rusty-brown sputum. After
violent exertion, and at times without manifest reason, attacks of haemoptysis of
varying degrees of severity come on. The characteristic ova are constant in the
sputum and establish the diagnosis. The fluke itself is a little more than Y$
inch (8 mm.) long and is almost round on transverse section, there being, however,
some flattening of the ventral surface. The acetabulum is conspicuous and opens
just anterior to the middle of the ventral surface. Eggs about 90 X 6s/z.

The branched testicles are posterior to the laterally placed uterus and the genital
pore opens below the acetabulum. The branched ovary is opposite the uterus on the
other side.

It is rather flesh-like in appearance and is covered with scale-like spines. The
flukes are usually found in tunnels in the lungs, the walls of which are of thickened
connective tissue. There may be also cysts formed from the breaking down of
adjacent tunnel walls. In addition to lung infection with this fluke, brain, liver,
and intestinal infections may be found. Musgrave was the first one to call attention
to the frequency of general infection with this parasite (paragonimiasis) in the

300

FLAT WORMS

J 4-U~

Philippines. He found it in 17 cases in one year. The life history, beyond the
the stage of miracidium, is unknown.

Another fluke which has been reported from the lung is Fasciola gigantea (very
similar to F. hepatica). This was coughed up by a French officer who had been in
Africa.

Ova of the Parasitic Worms or Man
TREMATODA

DRAWN TO S C A L C X IOOO

Heterophyes
heterophyes

Dicro
coelium
lance utum

P«»~*4 Fasciola

*_ —

Fasciolopsis

Opisthorchis
felineus (

Clqiiorcliis Clonorchis
sinensis endemicus

iMudiix-il fioiii Looss 1(K>;)

n i

//J;

Schistosoma

FIG. 66. — Trematode ova.

BLOOD FLUKES

Schistosoma haematobium. — Flukes of the circulatory system are
of great importance in Egypt, South Africa, Japan, and the West
Indies. The disease is named bilharziasis after Bilharz who in 1851
first associated the parasite and the disease.

It seems probable that there are at least three human species,
differentiated principally by the appearance of the egg. In the blood-
fluke disease of Egypt (S. hamatobium) , the parasite chiefly infects
the bladder and the egg has a terminal spine. The terminal-spined
ovum is also found in the rectum and in the faeces. In the West

BLOOD FLUKES

301

Indies, as shown by the reports of Surgeon Holcomb from Porto Rico,
rectal bilharziasis is rather common. In these cases the egg is prac-
tically always lateral-spined. Looss thinks that the lateral-spined egg
is the product of an unfertilized female S. hamatobium. These flukes
differ from other human flukes in possessing nonoperculated eggs as
well as in having the sexes separate. The adults of this species, the
S. mansoni, are scarcely, if at all, to be distinguished from the S.
hamatobium. Leiper has recently noted a difference in that the male of

FIG. 67. — Anatomy of a tape- worm, T&nia solium (A., longitudinal, B., cross
section); a fluke, Paragonimus westermanii (C)., male and female nematode, Oxyuris
vermicularis (D.). A. i, Testes; 2, yolk glands; 3, shell glands; 4, ovary; 5, vagina;

6, vas deferens; 7, uterus before branching; 8, water-vascular system. B. i, Cuticle;
2, circular muscle; 3, ovary; 4, testes; 5, uterus; 6, excretory canal; 7, nerve cord.
C. i, Oral sucker; 2, acetabulum; 3, uterus; 4, testes; 5, excretory canal; 6, ovary;

7, yolk glands. D. (a) Female, i, Vulva; 2, uterus; 3, bulb of oesophagus; 4, anus;
(D) Male. i. Bulbous mouth end; 2, testes; 3, spicule; 4, alimentary canal. E.
Egg. of P. westermanii.

S. mansoni has seven testicles as against four for S. hamatobium. With
S. japonicum, the name of the Eastern species, there is not only the
difference that the eggs are without spines, but, in addition, the skin
of the adult parasite is not tuberculated, as is the case with the other
two species. It is slightly smaller, the acetabulum projects more
prominently, and the lower part of the male infolds more markedly

302 FLAT WORMS

than in S. hcematobium. Catto considers that the S. japonicum may
live in both arteries and veins. The other two species only live in
branches of the portal vein. The blood flukes are about J£ inch (13
mm.) long. All of these flukes live separately until maturity. At
this time the female enters what is known as the gynaecophoric canal
of the male; this canal is formed by the infolding of the sides of the flat
male fluke, thus giving a rounded appearance to the male. The
female is longer than the male (about % inch long), and is thread-like
and of a darker color. Her two extremities project from the canal of
the male in which she lives.

The oral sucker of the male is infundibuliform and is smaller than the
pedunculated acetabulum. In the female the oral sucker is larger than
the acetabulum. The eggs of S. hcematobium are fusiform, yellowish in
color, have a thin shell and a terminal spine.

The most prominent symptoms of the Bilharz disease are haemat-
uria and bladder irritation; later on calculus formation.

In rectal bilharziasis the symptoms are more those of bleeding piles or of a mild
dysentery.

There may also be involvement of the appendix.

In the Japanese infection the symptoms point more to liver and
spleen, there being ascites, cachexia, and a bloody diarrhoea. Early
in the infection we have fever, urticarial spots and some bronchial
trouble (urticarial fever). The eggs should be searched for in the
mucus cap on the faeces.

The eggs of the S. japonicum are readily found in the faeces; they are about 100 X
7o/i. They are oval, transparent, and with a smooth shell, within which can be made
out the outlines of an embryo. Upon adding water the ciliated embryo begins to
show movement in about ten minutes and shortly afterward bursts out of the shell
and swims about actively. It is more melon-shaped than the miracidium of S.
h&matobium.

The exact life history is not known of any of these flukes. Looss conjectures that it
is probable that the miracidium enters the skin, not requiring an intermediary host.
Frequent experiments have failed to show any mollusk, etc., which attracted the
embryo. Evidence seems to show that those who are constantly wading about in the
water of the pools or the mud of the fields are the ones most subjected to infection.

Katsurada, by experiments with a cat and dog, has proved that infection will take
place through the shaved skin of an animal held in infected water — none of the water
being allowed to enter by mouth. Fully developed miracidia and the male and
female flukes were found in the portal vein. It is thought that further development
of the miracidia in the body may account for the heavy infection.

LIFE HISTORY OF SCHISTOSOMA

303

Recently Leiper has found cercariae showing the absence of a pharynx
(characteristic of the genus) in a Japanese mollusc. Such molluscs
were teased out in water and laboratory bred mice immersed therein.
One of these mice was killed a month later and adult schistosomes
were found in the portal vessels. Leiper has also found cercariae
showing absence of pharynx in four different species of molluscs in
Egypt. With such molluscs he was able to infect white rats and
other animals. He states that infection with these cercariee from the
mollusc host can bring about infection either by way of the mouth or
through the skin. Sodium bisulphate in a strength of i to 1000 killed
these cercariae almost immediately.

It would therefore seem proven that all human schistosome in-
fections take place following cercarial and not miracidial development.
As proof that S. hcematobium and S. mansoni are different species,
Leiper notes that mice infected by molluscs of the genus Bullinus
showed schistosomes with terminal spined eggs, the ovary lying in the
lower half of the female. The male had four or five large testes. In
mice infected by molluscs of the genus Planorbis, the eggs were lateral
spined, the ovary was in the anterior half of the body and the male

had eight small testicles.

•

There is a view that the miracidium enters while bathing by the preputial channel,
hence the value of circumcision.

If urine containing eggs is diluted with water the miracidium breaks out of the shell
and swims about as if in search of some desired object.

The view is also entertained that the miracidium may gain access to the body
through the drinking water; there is much evidence against this. However access
to the body is gained, it is known that the larval forms make their way to the liver
where they develop. Arriving at maturity, the males and females become united
and proceed to the terminal branches of the portal vein, where the irritating eggs,
given off by the female, give rise to the symptoms.

CESTODE OR TAPE-WORM INFECTIONS

The cestodes and trematodes constitute the two great divisions of
the flat worms. Anatomically, a tape-worm may be considered as a
series of individual flukes united in one ribbon-like colony. The
cestode segments, or proglottides are covered by an elastic cuticle and
in their interior usually contain striated elliptical bodies composed of
calcium carbonate about 5 to 25^ according to the species in which they
are found.

3°4

FLAT WORMS

These calcareous bodies are characteristic of cestode tissue. They have been
mistaken for coccidia. There is no mouth or alimentary canal in tape-worms, the
segments absorbing their nourishment through the general surface.

A tape- worm is divided into the segment-producing controlling head and the series
of segments or proglottides together known as the strobila. The head and neck
together form the scolex.

The head contains the central nervous tissue and the commencement
of the water-vascular system.

DibothriocephaliA latua

Taenia solium

Taenia saginata

FIG. 68. — Adult and larval stages of cestoda of man.

Tape-worm heads are provided with suctorial or hook-like organs, or both, to
enable them to hold on to the intestinal mucosa.

The hooks when present on the anterior extremity of the head are carried by a
protrusible structure called the rostellum.

The importance of the head is generally recognized by the well-
known fact that the permanent evacuation of one of these parasites is
only arrived at when the head as well as the segments is expelled.
Otherwise, additional segments will be produced.

Even in tape- worms 25 to 30 feet in length, the head is no larger than a small shot.
It carries the suckers or booklets which best enable us to differentiate the different

CE5TODE5 3°5

species. The segments adjacent to the head are immature — the sexually mature
ones being found from the middle of the body onward.

T. sagifmia has about 2000 segments, T. solium less than 1000 whfle T. eckino-
coccus has only three or four. The sexually mature segment possesses a varying
number ol testicles: three in Hymcnolepsis nana and as many as 2000 in Tcrnia
sagittate. As with the flukes, they also have vasa deferentia, cirrus, ovaries, yolk
glands, uterus, genital pore, etc. The location of the genital pore and the charac-
ter of the branching of the uterus are of the greatest importance in differentiation.
The sexually mature proglottides may either expel their ova, when these would be
found in the faeces or, as is common, they break off and pass out themselves in the
faeces. Then they either expel the eggs or may be eaten by some animal and in
this way effect an entrance for their ova. It is an important practical point that
the faeces of a patient with T. solium or T. sagittate may not show any ova, these
passing out in the intact segments. The oval operculated eggs of Diboikriocephalus
lotus, however, are constantly in the faeces.

The "hexacanth" or six-hooked embryo, also called the onchosphere, is the essen-
tial part of the egg. The embryonic envelope is dissolved off in the alimentary
canal of the animal ingesting it, and the onchosphere bores its way through the gut
to later become encysted in various tissues. In some tape- worms a ciliated embryo
is liberated from the egg shell and swims about actively to enter some fish or other
animal. When the six-hooked embryo reaches its proper tissue, the booklets are
discarded and a scolex similar to the parent one is developed. At this time we have
a bladder-like structure with the scolex inverted in it. This is termed the proscolex
stage. This Kttk cyst with its scolex when ingested by another animal is digested,
and the sccdfr, establishing itself in the intestine, develops a series of segments.
The ciliated embryo of the D. lotus does not form a cyst, but instead a worm-like
creature q'milar to the adult. This is termed a Plerocercoid.

If the larval stage shows a single cyst and a single head, it is termed
Cysticercus; if multiple cysts but only one head to each cyst, Ccenwrus;
while with multiple cysts and multiple heads hi each cyst the term
Eckinocvccus is used.

Where there is very little fluid in the cyst and the larva is of minute
as with the HymenoUpsis, the term Ccrcocystis is employed.

KEY TO CESTODE GENEEA

I. Head with two elongated sfit-Kke suckers— Genital pores ventral— Rosette
uterus. Dibothriocephabdae.

(A) Single set of genital organs in each segment. Dtbotkrioccpkalus.

(B) Double set of genital organs in each segment. Dipbgottoporus.

(O Immature forms showing characteristics of Dibothriocephalidae— (collective

group). Sparganum.
II. Head with four cup-like suckers; genital pores lateral Taeniidae.

(A) Uterus with "M^Kaii stem and a varying number of lateral branches. Tctnia.

(B) Uterus without median stem and lateral branches.

FLAT WORMS

(1) Genital pores single. Rostellum with not more than two rows of hooks,
(a) Suckers armed with numerous small booklets. Fifteen to twenty

testicles in each segment. Davainea.

(6) Suckers not armed. Three testicles in each segment. Hymeno-
lepsis.

(2) Genital pores double. Rostellum with four or five rows of hooks.

Dipylidium.

TJENIID^E INFECTIONS

Tsenia saginala (Taenia mediocanellata). — This very widely dis-
tributed tape-worm is often termed the unarmed tape-worm, to
distinguish it from the T. solium or armed tape- worm.

It is from 10 to 25 feet long and has several hundred proglottides. The small
pear-shaped head has four pigmented elliptical suckers and no booklets. The seg-
ments are plumper than those of T. solium, hence the name saginata. The single
lateral genital pore projects markedly and in a series of segments presents, as a rule,
first on one side, and then on the opposite side of the next segment (alternating).
The best way to distinguish a segment of the T. saginata from the T. solium is by
counting the number of lateral uterine branches; these number fifteen to thirty, are
quite delicate and branch dichotomously. The lateral divisions of the uterus of
the T. solium are tree-like in their branching and only number five to twelve on each
side.

T. solium has three ovaries while T. saginata has only two. The ox is the inter-
mediate host of T. saginata. The eggs of Tcsnia have an oval outer shell which is
filled with rather translucent, refractile yolk, often in globules. Within the oval
shell is the more rounded cell of the six-hooked embryo with its thick striated
membrane. The outer shell is often absent in the eggs found in the faeces, only
the shell of the six-hooked embryo being found. The six-hooked embryo, having
worked, its way from the alimentary canal to the muscles or liver of the ox,
becomes encysted (Cysticercus bovis). This little bladder-like structure is about
Yi by y$ inch, and contains but a small amount of fluid.

The evaginated head does not show booklets, they differing from the
armed rostellum of the scolex of Cysticercus cellulosce.

Being ingested by man's eating raw or imperfectly cooked meat, the adult stage
becomes established in his alimentary canal in about two months.

It is probable that the various raw-meat cures have made the infection more com-
mon. Cysticercus bovis is more abundant in the tongue of cattle than elsewhere in
the musculature.

For this reason it would seem advisable to use other raw meat than beef in such
cures.

In Abyssinia the infection is said to be universal, and a man without a tape-
worm to be a freak. An important point is the fact that the larval stage almost
never appears in man. It is this fact which makes it a so much less dangerous para-
site than the T. solium, which readily establishes a larval existence in man if the ova

HYMENOLEPIS 307

are introduced into the human stomach. Cooking meat always destroys the
cysticercus. A period of about two months elapses after the ingestion of the cysti-
cercus before the mature segments pass out of the rectum. These not only make
their exit with the faeces, but are also capable of wandering out at other times. In
this they differ from the segments of T. solium. T. saginata next to Hymenolepis
nana is the common tape-worm of the United States. Dr. Stiles has examined
several hundred tape-worms in the United States during the past few years and
has found only one T. solium.

In Paris Blanchard found 1000 T. saginata to 21 T. solium. Certain
German statistics, however, show about one-half as many T. solium
as T. saginata.

Abnormalities of the scolex and proglottides are not uncommon with T. saginata.
This is less frequently the case with T. solium.

Taenia solium. — The measly pork tape-worm is smaller than the
T. saginata and differs from it in having a globular head, with a ros-
tellum which is crowned by 26 to 28 booklets.

In T. saginata a depression takes the place of the armed rostellum; the suckers
of T. saginata are, however, much more powerful than those of T. solium. The
segments have only five to ten coarse branches and are expelled only at the time of
defecation. The segments or the ova having been ingested by a hog, the six-hooked
embryo is liberated and becomes encysted chiefly in the tongue, neck, and shoulder
muscles of the hog, as an invaginated scolex. Pork containing this cysticercus
(Cysticercus celluloses) is known as measly pork. This cysticercus contains much
more fluid than that of the ox and is from % to ^ inch long. If one by
chance should carry the egg on his fingers to his mouth, as the result of examining
mature segments, the larval stage may be established in man. If this infection is
not heavy, very few symptoms may be observed. The cysticercus, however, tends
to invade the brain, next in frequency the eye, and so causes convulsions, death or
blindness. Instead of only being the size of a pea, these cysts, when forming in the
brain, may be the size of a walnut or larger. T. solium is comparatively common in
north Germany, but is exceedingly rare in England and the United States.

Taenia africana. — This is an unarmed tape-worm, only about 5 feet long. It
was found in a native soldier in German East Africa.

Garrison has reported from the Philippines a tape-worm with an unarmed
rostellum, V-shape and spiral formation of the uterine stem with compact structure
of the gravid uterus under the name of Tania philippina. Another tape-worm,
T. confusa of which only segments were found was reported by Ward from Nebraska.

Hymenolepis nana (Tsenia nana). — This is generally known as the
dwarf tape- worm — it is the smallest of the human tape-worms. It is
from Y± inch to ^ inch in length, and is less than >^5 inch in breadth.
(10 X i mm.)

308

FLAT WORMS

The genus Hymenolepis has lateral genital pores, all of which are on the same
side. These lateral genital pores cannot be made out in specimens as ordinarily
examined. The head has four suckers and a rostellum, which is usually invaginated.
The rostellum has a single row of 24 to 30 booklets encircling it. Of the 150 to
200 narrow segments the terminal ones are packed with eggs which in the last two
or three seem to fill entirely the disintegrating segments. It would seem that the
fully mature segments disintegrate and in this way the eggs are set free in the
surrounding intestinal contents.

The worms as found in fresh faeces after taeniacide treatment are frequently in an

a- Laminated elastic cuticle
b • fmer yermmal layer
c - Scoitr frte In cyst
d- Brood capsule

;- Endogenous daughter cyst
- RufJurvd brood capsule

-b.

HYMENOLEPIS
DIMINUTA.

FIG. 69. — Other cestodes of man.

advanced state of disintegration so that it is impossible to make out the head or
booklets.

The eggs of this species are quite characteristic, there being two distinct mem-
branes. The inner one has two distinct knobs, from which thread-like filaments
proceed. The eggs of the H. diminuta have a thicker, striated, outer membrane
and there are no filaments. The eggs of the Dipylidium caninum are similar, but are
found in the faeces in aggregations — several eggs in a packet.

The dwarf tape-worm has been found to be the most common tape-
worm in the United States. Dr. Stiles found it in about 5% of children
in a Washington orphanage.

DIBOTHRIOCEPHALUS

309

It has been estimated that in certain parts of Italy 10% of the children may be
infected. The symptoms, especially nervous ones, may be marked in this infection.
It has been incriminated as a cause of chyluria. Although very small, yet the num-
ber of parasites may be very great, even more than 1000. In a case that I treated
with thymol there were 1500 worms expelled. A form found in rats, which may be
identical with H. nana, does not require an intermediate host. The six-hooked
embryo bores into the intestinal villus and there develops a Cercocystis (larva of
small dimensions with but little fluid). When fully developed, it drops into the
lumen of the gut, and a new parasite is added to the already existing number of
parasites. This explains the heavy infection. H. diminuta and H. lanceolata have
also been reported for man a few times.

H. diminuta is much larger than H. nana, being about 10 inches long. The
suckers are small and the rostellum insignificant and unarmed. The intermediate
host in some insect, as a moth; the definitive, the rat. As man is not liable to eat
the insect hosts the infection is rare in man. Twelve cases have been reported for
man of which five were from the U. S.

H. lanceolata is common in geese and ducks.

Dipylidium canimim (Taenia cucumerina) (T. flavopunctata). — This is a common
parasite of dogs and cats. The larval stage is passed in lice and fleas. The cases of
human infection have been principally in children, probably from getting dog lice
or fleas in their mouths. The number of infections reported for man is about 40
and of these about 30 in children. The head has four suckers and a rostellum,
which has three or four rows of encircling booklets. The segments have the shape
of melon seeds and have bilateral genital pores.

Davainea madagascariensis. — This tape-worm has been found in Siam and
Mauritius. It is about 10 inches long. The head has four suckers and a rostellum
with 90 booklets. The suckers have rings of booklets. The genital pores are
unilateral. The cockroach is supposed to be the intermediate host.

There have been about 10 cases reported (Madagascar, Siam and British Guiana).
There has also been reported a D. asiatica, the single specimen, however, lacking a
head so that the exact genus is doubtful. It has been reported twice in children in
Breslau. The intermediate host is thought to be a cyclops. Garrison reported
cases from the Philippines.

DlBOTHRIOCEPHALID^E INFECTIONS

Dibothriocephalus latus (Bothriocephalus latus). — This is fre-
quently termed the broad Russian tape-worm. It has a small olive-
shaped head with two deep winding suctorial grooves on each side; it
has neither rostellum nor hooklets.

The segments are quite broad, being about /^ by 3^ inch. At the end of the
strobila they are more nearly square. The segments are very numerous, 3000 or
more. The fully developed worm is about 30 feet long. The uterus in each segment
is rosette-shaped and the genital pore is ventrally situated. The eggs of this species
have an operculum and a ciliated embryo. This ciliated embryo swims around and
either enters some fish, especially pike, directly or through an as yet unknown inter-

3io

FLAT WORMS

mediary. This parasite produces an intense anaemia similar to pernicious anaemia.
It is a frequent parasite in Switzerland, Bavaria, Japan, Scandinavia, and Russia.
Recently several cases have been reported from our Northwest, and some of the fish
of the waters of that region are said to be infected. The larva is a pleroceroid and
is about i inch long. It is said that salting, smoking, or other ordinary methods of
preserving fish will not kill it.

A tape-worm, Diplogonoporus grandis has been reported from Japan. In this
there are two complete sets of genital organs to each segment.

SOMATIC T^NIASIS

While rarely we may have the larval stage of T. solium present in
man, and while certain bothriocephalid larvae (Sparganum mansoni
and Sparganum proliferum) infect man, yet they are unimportant as
compared with the larval stage of the Tcenia echinococcus.

FIG. 70, — Daughter cyst from hydatid
cyst, considerably enlarged. (Coplin.)

FIG. 71. — A group of daughter cysts
from hydatid cysts. (Coplin.)

Taenia echinococcus. — The adult stage of this parasite is passed in
dogs. It is one of the smallest tape-worms known, being only about
3^5 inch long. It has a head with four suckers and a rostellum en-
circled with hooks. There are only three to four segments. The larval
stage, on the contrary, gives one of the largest of larval cestodes. In
man it may reach the size of a child's head.

The larval stage is also found in hogs and sheep, and it is probable that by reason
of the dog's eating the echinococcus cyst of such animals at the abattoir we owe
the increase in this serious infection.

Man contracts the infection from association with dogs. The disease is peculiarly
prevalent in Iceland. As stated above, the adult stage is passed in the intestine of

HYDATIDS

the dog. Should the egg-bearing segments passed by the dog contaminate the hands
of man and a single egg be ingested, we may have hundreds of Tania larvae produced.
The six-hooked embryo, leaving its shell, bores its way through the walls of the
alimentary tract and especially seeks the liver, just as the embryo of T. solium seeks
the brain and eye.

Griffith notes that in Australia from 10 to 15% of hydatid cysts
occur in the lungs. The cyst wall is quite thin and the hydatid
cachexia seems to appear earlier in the lung than in the liver cases.

Ova or the Parasitic Worms or Man
CESTODA

ORAWM TO SCALE X IGOO

i^iuiu- ^*=^- Dibothrio- — - — -

gonoporus cephalus nymenojepisimna

Hymenolepi

^ diminuta

Toenia^Bf

vta^ \^,^'r

Dipylidium

Cestode segments

DRAWN TO SCALE X IO O

Da\5in / U^l

m^^?^SFiensis H^no- /

lepis /« l**?^*

FIG. 72. — Cestode ova.

In the development of the cyst, after the embryo has come to rest at some point
in the liver, we have formed at first an indistinctly laminated external envelope
with coarsely granular fluid contents. Later on the contents become transparent,
and two distinct layers can be observed: i. The external, markedly laminated one,
and 2. the internal one, made up of small cells externally and large cells and cal-
careous corpuscles internally. This internal lining membrane is known as the paren-
chymatous or germinal layer. When the external layer is incised it curls up by
reason of its elasticity. This is characteristic of such a cyst. In addition, we have
an enveloping connective-tissue capsule formed by the surrounding liver substance.

312 FLAT WORMS

From the germinal layer arise the brood capsules and the scolices. In these brood
capsules we have the cellular layer external — just the reverse of the mother cyst.
Scolices may develop either on the outside or inside of these brood capsules. It is
interesting to note that one onchosphere may develop hundreds of scolices. From
the parenchymatous layer of the mother cyst, daughter cysts are formed; these have
an external stratified layer and an internal parenchymatous one; within them a vary-
ing number of scolices may develop. From these daughter cysts, granddaughter
cysts may arise — all within the mother cyst — and hence are termed endogenous.

At times the daughter cysts work their way external to the mother cyst and pro-
ceed to develop in a manner similar to the endogenous formation. The exogenous
development is rare in man, but common in hogs. Hydatids containing no scolices
are called sterile. These cysts may be as large as a child's head, but are usually
smaller. The fluid of these cysts contains about i% of NaCl, also a trace of sugar;
in addition there is a toxin which produces urticaria and acts as a cardiac depressant.
If any quantity should escape into the peritoneal cavity at operation, it may cause
death. Hydatids develop very slowly, and the duration of the disease is usually
from two to eight years.

Echinococcus multilocularis is possibly due to a species different from T. echino-
coccus. In this we have a honeycomb arrangement with cavities filled with a gela-
tinous material. The majority of these cysts are without scolices. This form of
hydatid is very fatal.

Sparganum mansoni (Bothriocephalus liguloides). — This is a larval bothrio-
cephalid which is about 5 to 10 inches long and has been reported 10 times in Japan.
It has been found in various parts of the body, as in pleural cavity, tissues about
kidney, and in abscess of the thigh. They have been found in the urethra and under
the conjunctiva. They resemble ribbon-like strings of fat.

Sparganum prolifer (Plerocercoides prolifer).— This has been reported from
Japan as a larval form in the subcutaneous tissue. Stiles has found these larval
forms in skin lesions in Florida. They show themselves as bizarre grub-like forms.
They reproduce by budding.

CHAPTER XVIII
THE ROUND WORMS

CLASSIFICATION OF THE NEMATHELMINTHES (ROUND WORMS)

Class

Nematoda

Family
Angiostomidae

Filariidae

Trichotrachelidae

Strongylidae

Ascaridae

Genus

Species

Strongyloides

S. stercoralis.

Dracunculus

D. medinensis

F. bancrofti

F. loa

F. perstans

Filaria

F. demarquayi

F. ozzardi

F. philippinensis

Onchocerca

O. volvulus

f Trichuris

T. trichiura

1 Trichinella

T. spiralis

Eustrongylus

E. gigas

Strongylus

S. apri

Trichostrongylus

T. instabilis

Triodontophorus

T. deminutus

(Esophagostoma

O. brumpti

Physaloptera

P. caucasica

Ancylostoma

A. duodenale

Necator

N. americanus

Ascaris

A. lumbricoides

Belascaris

B. mystax

Oxyuris

O. vermicularis

Gigantorhynchus

G. gigas

' Hirudo

H. medicinalis

Limnatis

L. nilotica

Haemadipsa

H. ceylonica

Acanthocephali

Hirudinei

NOTE. — The Strongyloides stercoralis was formerly described under two desig-
nations: (i) Anguillula inteslinalis, a parasitic generation and (2) Anguillula ster-
coralis, a free living generation.

ROUND WORMS OR NEMATODES

All nematodes are covered by a cuticle which varies in thickness,
and is frequently ringed. The cuticle is moulted three or four times.

313

THE ROUND WORMS

rule.

The cuticle is formed by the underlying ectoderm which is, as a rule,
markedly developed in four ridges which divide the body into quad-
rants. Within the ectoderm is the body cavity, a space in which the
reproductive organs lie in a clear fluid. The excretory system usually
consists of two tubes which discharge near the head.

While the alimentary canal is more or less tube like in appearance it shows near
the mouth a muscular oesophagus with a bulb-like expansion at the commencement
of the remainder of the intestinal tract. There is a nerve ring around the oesophagus.

The testis and ovary are generally tube like. The sexes are, as a rule, separate.
The male can usually be recognized by its smaller size, its curved or curled posterior
end, and at times exhibiting an umbrella-like expansion — the copulatory bursa.
The spicules, chitinous copulatory structures, may be observed drawn up in the worm
or projected out of the cloaca. The genital opening of the female is ventral and
usually about the mid-point; that of the male is close to the anus.

Certain papillae in the region of the anus are valuable in differentiation. As a
rule, nematodes develop in damp earth from the eggs as rhabditiform larvae. Very
few nematodes are viviparous (Filaria, Trichinella) being usually oviparous (As-
caris) less frequently ovo viviparous (Oxyuris).

The families Gnathostomidae and Anguillulidae are of very little
importance in human parasitology. Gnathostoma siamense was once
found in a breast tumor and Rhabditis pellio once in the urine.

Anguillula aceti, the vinegar eel, has been reported from the genito-urinary tract
several times. Such cases can be explained by the prior contamination of the urine
bottle or by the use on the part of the patient of a vinegar vaginal douche. The
genera Rhabditis and Anguillula belong to the family Anguillulidae.

A case of infection with a small nematode found in the papules of a skin infection,
in a French boy is recorded as due to Rhabditis niellyi. The present view is that
the parasites were embryos of A. duodenale, boring into the skin.

ANGIOSTOMID,E

In this family we have heterogenesis.

Strongyloides stercoralis. — This parasite was formerly thought
to be the cause of Cochin-China diarrhoea. It presents two genera-
tions: i. Parasitical or intestinal form. 2. The free living or faecal
form.

i. The intestinal form (also known as Anguillula intestinaHs} is represented only
by females. These are about ^2 inch (2 mm.) long and reproduce partheno-
genetically. They have a pointed, four-lipped mouth, and a filariform oesophagus
which extends along the anterior fourth of the body. The anus is situated near the
sharpened posterior end, the vulva about the lower third of the body. The uterus
contains a row of eight to 10 elliptical eggs which stand out prominently in the pos-

THE GUINEA WORM 315

terior part of the body by reason of being almost as wide as the parent worm. They
usually live deep in the mucosa and the embryos emerge from the ova laid in the
mucosa. The embryos escape from the eggs while still in the intestine, so that in
the faeces we only find actively motile embryos. The eggs, which are strung out
in a chain, never appear in the fasces except during purgation. As they greatly
resemble hookworm eggs, this is a point of great practical importance. In fresh
faeces we find hookworm eggs and Strongyloides embryos. The embryos are rather
common in stools in the tropics. These embryos have pointed tails and are about
250 X i3M- They have a double cesophageal bulb. They are about 250*1 when they
first emerge but may grow until they will approximate 500/1 in the fseces. If the
temperature is low, these rhabditiform embryos develop into filariform embryos,
which being ingested form the infecting stage. It has been demonstrated that
infection of man may also take place through the skin. If the temperature is warm,
25° to 35°C., these embryos develop into:

2. The free living form, Anguillula stercoralis. In this we have males and females,
with double cesophageal bulbs, the male about ^0 inch (% mm.) long with an in-
curved tail and two spicules and the female about %5 inch (i mm.) long with an
attenuated tail; these copulate and we have produced rhabditiform larvae, which
later change to filariform ones. At this time the length is about 550 microns.
These, being ingested, start up the parasitical generation. If these do not reach the
intestine they die out.

FILARIID.E

This family is of the greatest importance to man. It is also one
about which much confusion exists as to the adult type; hence anyone
finding adult filariae should fix them in hot 5% glycerine alcohol
(alcohol 70%), and subsequently mount in glycerine gelatin. Formalin
is not to be used, other than for a very brief period (two to six hours)
and then followed by the lacto-phenol method.

These worms are most likely to be seen as writhing thread-like worms, especially
in the lymphatic glands and connective tissue, and about body cavities. They have
a lipped or simple mouth and a filariform oesophagus. The male has an incurved
tail with preanal and postanal papillae which may be even corkscrew-like as in F.
immitis. The spicules are unequal or there may be but one. The female is ovovivi-
parous, the vulva is at the anterior end and the uterus usually double.

Dracunculus medinensis (Filaria medinensis). — The Guinea or
Medina worm, of which until recently only the female was known, is
of great importance in parts of India, Africa, and Arabia. The female
is a thread-like worm, about 20 to 30 inches long. The habitat is the
subcutaneous and intermuscular connective tissue, especially of the
lower extremity. It develops without symptoms. Finally a blister-
like area appears on the surface of the leg, particularly about ankle-
joint, which soon forms a painful ulcer. From this opening the

THE ROUND WORMS

ryos

anterior end of the worm projects to pour forth its striated embry
upon contact with water.

The mouth is terminal and the body uniformly cylindrical. The uterus is a con-
tinuous tube filled with sharp-tailed, transversely striated embryos, 650 X i7/i, and

Fig. 73. — (la) Adult female Guinea worm (Dracuncidus medinensis} showing
anchoring hook at posterior extremity, (ib) Cross section of female Dracunculus
showing uterus filled with embryos, (ic) Striated embryos of the Guinea worm,
(id) Cyclops coronatus, the minute crustacean which serves as the intermediate host
of D. medinensis. (2a-2d) Anterior and posterior extremities of F. loa. (zc) Sec-
tion showing tuberculated cuticle. (2b), Male and female F. loa, natural size. (3a)
Bulbous anterior extremity, Filaria bancroft. (36) Tail of male. (3c) Tail of female.
(3d). Male and female, natural size of F. bancrofti. (4a) Tumor mass of F. volvulus
laid open. 5, Mosquito showing filarial embryos in thoracic muscles (a) and in
labium (b). The labella which are separated from the labium by Button's mem-
brane are seen at (c). 6. (a) Embryo of F. bancrofti (b) embryo of F. loa showing
filling of tail end with cells. 7, Micrafilaria of F. bancrofti in blood. Dotted lines
show location in break in cells column and V spot.

constitutes the greater part of the body, the alimentary canal being pressed to one
side. The genital organs probably discharge through the oesophagus. The body
when being extracted is rather transparent. The tip of the tail is bent, forming a
sort of anchoring hook. Recently Leiper fed monkeys on bananas containing in-
fected Cyclops, and at the autopsy six months later obtained both male and fe-
male forms.

FILARIAL WORMS 317

As regards the life history, Fedschenko, in 1870, showed that the embryos when
liberated swam around in water and finally entered the bodies of species of the genus
Cyclops. The female tends to come to the surface in the lower extremities, and
experiments show that if on the blister-like points of emergence some water be
squeezed out from a sponge, the uterus will eject a milky-looking fluid containing
myriads of embryos. This would indicate that the worm selects the lower extremity
so that the embryos may gain access to the Cyclops when the host is wading through
the water.

Leiper showed that a strength of HC1 equal to that of gastric juice killed the
Cyclops, but made the Dracunculus embryos very active. From this he judged that
infection must probably take place from drinking water containing infected Cyclops.
The suggestion of Leiper that wells harboring Cyclops be treated with steam, intro-
duced by a pipe, seems to be valuable. The disease is known as "Dracontiasis."

Filaria loa (Filaria oculi). — This is a thread-like worm of west
Africa about i to 2 inches long. The cuticle is characterized by
distinct wart-like structures.

The anterior extremity is like a truncated cone with two papillae at the base of
the cone. The wart-like cuticular protuberances or bosses are about 12 to 15
microns in height. The females are 2 to 3 inches (50 to 70 mm.) long and about %
mm. broad.

The males are smaller than the females and have three preanal papillae and two
postanal ones. There are two short unequal spicules. The life history is not satis-
factorily established. The young are born ovoviviparously, and it has been sug-
gested that the localized cedemas, known as Calabar swelling, may be due to the
irritation produced by these eggs. These swellings are of hen's egg size, painless,
do not pit on pressure and last about three days. They occur especially on the hands
and arms. The embryos almost exactly resemble those of F. bancrofti. They have
a diurnal periodicity, however, appearing in the blood about 8 A. M., increasing to
noon and disappearing about 9 p. M. The adult worms have a tendency to wander
about in the subcutaneous connective tissue, especially about the region of the orbit
or even under the conjunctiva.

Adult worms of F. loa have been found and extracted, with an absence of the
filarial embryos in the peripheral circulation of the patient. While immature adult
worms have been extracted from children the embryos have only exceptionally been
found in these children. This would speak for a very long developmental period
for the adult worm and as a matter of fact the infection often only shows itself years
after the opportunity for infection.

Leiper has just noted two species of Chrysops (Mangrove Flies) as intermediate
hosts, the embryos developing in the salivary glands.

Filaria bancrofti (Filaria sanguinis hominis). — This is the most
important of the filarial worms. It is a common infection in south
China, India, the West Indies, and in the Pacific Islands, especially
Samoa.

318 THE ROUND WORMS

f -I -M -I C

In medical books the embryos have been designated Filaria sanguinis hominis.
This species is the cause of the common manifestations of filariasis, such as elephanti-
asis, varicose groin glands, chyluria, lymph scrotum, etc.

Filarial diseases are prone to lymphangitis attacks. Thus in lymph scrotum an
erysipelatoid condition of the scrotum with high fever and chills may result. This
condition is at times mistaken for malaria. Varicose groin glands may be mistaken
for hernia. In the Philippines very few symptoms are noted in those affected with
filariasis. Occasionally chylocele or chyluria is reported.

F. bancrofti lives in lymphatics of trunk and extremities. At times the fine white
thread-like worms may be seen as writhing coils in lymphatic glands.

The sexes are usually found together. The females are about 3 inches long
and the males less than 2 inches. The tails of both sexes are incurved, but that of
the male is more so. The head is club-shaped. The vulva opens 1.2 mm. from the
anterior end. There are two uterine tubules. The sheathed embryos are supposed
to be born viviparously and Manson supposes that as a result of injury to the
parent worm and resulting extrusion of eggs, the blocking of lymph channels occurs.

A very interesting fact is that people with elephantiasis fail to show larvae in the
peripheral circulation. Manson considers that it is due to the blocking of the
lymph channels.

These embryos show a nocturnal periodicity. During the day they
remain in the lungs, and larger arteries.

If the patient sleeps in the daytime and is active at night the nocturnal perio-
dicity or presence of embryos in peripheral circulation is inverted. In the case of
F. loa, however, a change of habits does not change the periodicity of the filarial
embryos, they continue to appear in the peripheral circulation by day even if the
patient sleeps at that time.

The disease is transmitted especially by Culex fatigans. The sheathed embryos,
getting into stomach of mosquito, wriggle out of the sheath, they then bore their
way through walls of stomach and enter into a sort of passive stage, during which
further development takes place. They finally become distributed in the muscles
of the thorax and make their way along the fleshy labium, to enter the wound in
a person bitten by a mosquito, by way of Button's membrane. This development
takes about twenty days at which time the larvae are about }/\§ inch long and have
an alimentary canal. It was formerly considered that the filarial worm of the
Philippines was a different species, this from a study of microfilariae (F. phil-
ippinensis). Others, however, have considered the microfilariae as identical with
F. bancrofti. Recently Walker has published drawings and descriptions of four
adult filar iae in the Philippines which correspond to F. bancrofti.

Filaria perstans. — The adults are found in connective tissue and deeper fat,
especially about the mesentery and abdominal aorta.

The female is about 3 inches (75 mm.) long; the male is rarely found and is less
than 2 inches long. These worms are characterized by incurved tails, the extremity
of which has two triangular appendages giving a bifid appearance. The embryos do
not possess a sheath and have a blunt tail. The life history is unknown. Both
mosquito and tick have been incriminated. The embryos are always present in

MICROFILARIAE 319

the peripheral circulation — hence perstans. There does not seem to be any symp-
tomatology.

It is of historical interest that F. perstans was once considered the cause of sleeping
sickness.

Onchocerca volvulus (Filaria volvulus).— This is a rather common parasite of
central Africa. The male is about i% inches (35 mm.) and the female about 5 inches
long. The females are so interlaced in the fibro-cystic swellings that it is difficult to
determine their length. The tumors start from the presence of a worm in a lym-
phatic. The tumors are easily enucleated. Adults are striated. They are found in
cystic tumors, especially about the axilla and popliteal space. The cystic contents
contain abundant sheathless larvae about 300/1 long. It was formerly thought that
these larvae were absent from the peripheral circulation but more recent investiga-
tions have shown a sheathless embryo in the blood of patients with Onchocerea
nodules which had the characteristics of those found in the contents of the nodules.
Life history unknown, although it has been suggested that a species of Glossina may
be concerned.

Filaria demarquayi. — The habitat of this filarial worm is the West
Indies. The embryo has no sheath and has a sharp tail. Other filarial
species which have been reported are F. magalhcesi, F. ozzardi, F.
volvulus, F. powellij and F. philippinensis. A species called F. gigas
is now considered to have been only the hair of the leg of a fly. The
embryos have usually been given such names as F. nocturna, F. diurna,
etc. Of course the embryos and the parent should have the same name.
It has been proposed to designate these embryos the same as the parent,
but with the use of the term Microfilaria instead of Filaria.

The points usually noted in the description of filarial embryos are:

1. Presence or absence 6f periodicity of embryos in peripheral
circulation.

2. Presence or absence of a sac sheath around the embryo.

3. Accurate measurements.

4. Shape and description of head and tail ends.

5. Character of movement.

6. Location of V spot and break in cell column in stained
specimens.

KEY TO FILARIAL LARV.E IN PERIPHERAL CIRCULATION

A . Sheath present.

1. No periodicity.

F. philippinensis. Tightly fitting sheath; not flattened out beyond extremi-
ties. Tail is pointed and abruptly attenuated. Lashing progression move-
ment. 320 X 6.5;*.

2. Periodicity exhibited.

320

THE ROUND WORMS

(a) Nocturnal periodicity.

F. bancrof ti (F. nocturna) . Pointed tail ; loose sheath ; lashing movement.
300 X 7.5M- V spot gon from head; break in cells 50^ from head.

(b) Diurnal periodicity.

F. loa (F. diurna). Pointed tail; loose sheath; 245 by 7 microns. V spot
60 to 70 microns from head, break in cells 40 microns from head.

GENERAL FILARIAL KEY

Adults

Embryos

Remarks

Filaria
bancrofti.

Male 40 by o.i mm.
Female 90 by 0.28 mm.
Smooth cuticle. Bulb-
ous anterior extremity.
Occupy lymphatic
glands and vessels.

Sheathed, 300 by 7.5
microns. Distance from
head to V. spot 90 microns;
to break in cells 50 microns.
Tail rather straight. Ter-
minal cells do not fill up tail
end. Nocturnal periodicity
in peripheral circulation.

Transmitted by mos-
quitoes. Culex fati-
gans and Stegomyia
p s e u d o scutellaris.
Causes elephantiasis,
lymph scrotum, chy-
luria, etc.

Filaria loa. <

Male 27 by 0.3 mm.
Female 55 by 0.4 mm.
Cuticle tuberculated.
Anterior extremity like
truncated cone. Wan-
ders in subcutaneous
tissues.

Sheathed, 240 X 7 microns.
Distance from head to V
spot 65 microns; to break
in cells 40 microns. Cork-
screw tail which is com-
pletely filled up with ter-
minal cells. Diurnal
periodicity in peripheral
circulation.

Transmitted by
species of a bit-
ing fly — Chrysops.
Causes calabar swell-
ings. Worms often
visit ocular region.

Filaria
perstans.

Male 40 by 0.07 mm.
Female 75 by o.i mm.
Cuticle smooth. Tip
of tail shows two tri-
angular processes.
Found about root of
mesentery.

Without sheaths, 200 by 5
microns. Posterior two-
thirds tapers to blunt end-
ing. Distance from head
to V spot 49 microns; to
break in cells 34 microns.
Persists in circulation both
day and night.

Transmitting agent
not surely known.
Mosquitoes and ticks
suggested. No patho-
genicity.

Filaria
volvulus.

Male 30 by 0.14 mm.
Female usually frag-
mented. Possibly 75
by 0.36 mm. Cuticle
striated. Found coiled
up in cyst-like tumors
under skin.

Without sheaths. 250 by
7.5 microns. Found in
cyst-like spaces of tumors.
Found also in peripheral
blood and lymph glands.

Method of transmis-
s i o n unknown.
Causes small cystic
tumors, under skin of
thorax especially.

Filaria
medinensis.

Male from Leiper's
monkey 22 mm. Fe-
male 80 to 90 cm. long
by 1.6 mm. wide.
Smooth white body.
Anchoring hook at tail
end. Female lives in
subcutaneous tissue of
lower extremity.

Without sheaths. 600 X
20 microns. Long slender
tail. Cuticle striated. Ex-
truded from break in skin
of patient.

Embryos swallowed
by Cyclops. Man
drinks water con-
taining Cyclops.

THE WHIP WORM

321

B. Absence of sheath. None of these exhibit a periodicity, being continuously pre-
sent.

1. Blunt tail — F. perstans. 200 X 4.5;*.

2. Sharply pointed tail:

(a) F. demarquayi. 210 X SM-

(b) F. ozzardi. 215 X SM-

(c) O. volvulus. 250 — 3oo/i X 7-5M-

NOTE. — A filarial embryo, F. powelli, reported once. It has a sheath, nocturnal
-periodicity, and is about 130 X SM-

TRICHOTRACHELID^E

These have a long thin neck and a thicker terminal portion. The
oesophagus is of the single row of cells type. The anus is terminal;
there is only one ovary.

Trichuris trichiura (Trichocephalus dispar).— This is usually called
the whip-worm — the thickened body representing the handle and the
narrow neck the lash. It is one of the most common parasites in both
temperate and tropical climates.

The egg is very characteristic in having an oval shape with knobs at either extrem-
ity. It resembles a platter with handles. The male is almost 2 inches long, and has
the terminal portion curled up in a spiral. It has a single terminal spicule.

The female is a little longer than the male, and has the terminal part in the shape
of a comma instead of being coiled. The neck only contains the oesophagus which
is contained in a groove in large cells which form a single row like a string of pearls.
These cells play a digestion role. The vulva opens at the upper end of the thickened
terminal end which contains an intestine lying between the ovary and uterus. The
great powers of resistance of the ova may account for their general distribution;
they may live for months under conditions of freezing and so forth. There is no
intermediate host. The worm arrives at sexual maturity in about one month after
ingestion. The whip-worm prefers the caecum, but also lives in the lower end of the
ileum and the appendix.

The neck burrows into the mucosa, and much importance has been attributed by
the French to the possibility of this paving a way for the entrance of pathogenic
bacteria. They do not seem to produce serious symptoms.

Trichinella spiralis (Trichina spiralis). — The cause of trichinosis is
usually termed Trichina spiralis in medical works.

The adults live in the duodenum and jejunum; the males are about KG incn
(1.5 mm.) long with two tongue-like caudal appendages and without a spicule.
These two lateral projections enable the male to hold the female in copulation —
the cloaca being evaginated to act as a penis.

The females are about M inch (3 to 4 mm.) long. The female gives off
embryos from the vulva which is near the mouth end (viviparous).

322

THE ROUND WORMS

These parasites can be seen with an ordinary magnifying glass. With higher
powers the oesophagus has the appearance of a serrated line instead of an cesophageal
bulb. The male is about 40;* broad and has a prominent testicular enlargement
filling the posterior extremity. The female is about 6opt broad and has a rounded
posterior extremity with a prominent slit-like cloaca. It is in this posterior extrem-
ity that the female increases in size as she becomes filled with eggs. The vulva is in
the anterior third. After fertilization of the females the males die, and the females
bore into the intestinal mucosa and begin to produce embryos to the number of
more than 1000 each. These gain access to the lymph channels and are distributed

2. Stronyytoides etercoralia. (parasitic.)

1. Trichinella spiralis. A-Mhenooenetic female

B-Rhabditifirm embryo ,

7- Tern idena '£i. b-femole c-poftericr
demlnutuS nirtmity of mate, (spiiules)
d*e- anterior extremities.

brumpti

FIG. 74. — Some of the human nematodes.

by the blood-stream to the striated muscles. Embryos reaching other tissues fail to
develop.

It is about ten days before they reach the muscle. In the muscle they become
encysted as the oval lemon-shaped areas containing coiled-up embryos that every-
one is familiar with. These oval areas are about 450 X 250/1 and have a chitinous
capsule.

The encysted trichinae are found chiefly in the muscle fibers of the tongue and
diaphragm and may remain alive as long as ten to twenty years; finally, however,
the cyst undergoes calcareous infiltration and the embryo dies.

When uncoiled the embryo is about i mm. long with the mouth at the attenu-
ated end. Among cannibals it would be easy to keep the cycle going by eating
improperly cooked or raw human meat, the parasite being thus transmitted.

TRICHINOSIS

323

As this would not explain the transmission among civilized men, the
following is the life history: Man obtains his infection from eating raw
pork, the embryos encysted in the muscle of the hog being liberated in
the stomach, and the males and females developing in the intestine as
above described. The hog may gain his infection by eating the meat
of other hogs or rats. These rats eat scraps of pork at slaughter houses
a»d become infected. Being cannibals, rats when once infected, con-
tinue to propagate the infection. In man, during the first two or three
days while the adults are breeding in the intestine, we have gastroin-
testinal symptoms.

It is during this period or at any rate before the fifth day that purging may be of
benefit. About ten to twenty days after infection the embryos begin to wander and

FIG. 75.— Trichina spiralis. (Ziegler.)

we have the acute muscle pains. In the diagnosis we should try to obtain specimens
of the pork which has caused the trouble in order to examine for encysted trichinae,
or to feed to white rats or rabbits, subsequently examining the diaphragm of these
animals for encysted trichince or the intestine for adult trichinae. Excision of a
small piece of the deltoid of man may confirm the diagnosis. The best method is to
take blood in 3% acetic acid, centrifuge, and examine for larvae.

During the diarrhceal stage we may examine the stools for adult worms, in particu-
lar dead males or possibly actively motile embryos— these latter are about 90 X 6/i.

Always examine the blood for eosinophilia.

It is well to remember that the parts of meat which trichinae prefer (muscle of
diaphragm, of neck, etc.) are often used in sausage. Unfortunately it is almost
impossible to detect the embryos in sausage meat.

324 THE ROUND WORMS

STRONGYLID.E

In this family the male has a caudal bursa, a prehensile sort of ex-
pansion at the posterior end for copulatory purposes.

The mouth is usually provided with six papillae and at times with a
chitinous armature. Those without the chitinous armature are in-
cluded in the subfamily Strongylinae (Strongylus, Trichostrongylus)
while those having an armed mouth are in the subfamily Sclerosto-
minae (Ancylostoma, Necator, Triodontophorus, (Esophagostoma, Phy-
saloptera).

Eustrongylus gigas (Strongylus renalis). — This is the largest round
worm infecting man; it is usually found in the pelvis of the kidney
(giant strongyle).

•

Two or more worms may so distend the kidney as to convert it into a mere shell.
Pain, haematuria and other symptoms of pyelitis, together with the finding of the
eggs, make the diagnosis. There seem to be seven authentic and eight doubtful
cases of infection in man.

The females are about 40 inches (i m.) long and about Y$ inch (8 mm.) in
breadth while the male is about 10 inches (25 cm.) long.

The collar-like copulatory bursa of the male distinguishes it from Ascaris as does
also the dark red color. The source of infection is unknown but it has been suggested
that the larval stage may exist in fish.

Many of the reported cases were simply fibrinous clots from ureters or wandering
round worms.

The very characteristic ova, with gouged-out oval depressions, may be found in
the urine, and are diagnostically confirmatory.

Strongylus apri. — This nematode is common in the lungs of hogs, producing a
bronchitis in young animals but apparently harmless for adult ones. It has been
reported once from the lungs of a six-year-old boy. The male is about i inch (25
mm.) long with two long spicules. The female is about 2 inches long and has a
sharply hooked posterior extremity with the vulva just beyond the bend. The
mouth has six lips. The eggs contain embryos when laid.

Trichostrongylus instabilis. — This is a small strongyle formerly known as Stron-
gylus subtilis. The male is about % inch (4 mm.) long, and the female about
Y± inch (6 mm.) Anteriorly it tapers to a pointed head end which is only
about one-tenth the thickness of the posterior extremity. The male has a bursa
and two prominent equal spicules. It has been found in the upper part of the small
intestine of inhabitants of Egypt and Japan. It does not appear to produce symp-
toms. Ova like hookworm ones (73 to 90 X 42;*). Ransom gives T. col ubr if or mis
as the proper name. It is a common parasite of sheep and goats in the U. S. and
may exist in man in such regions. Stiles has kept in mind the possibility of such
infections in his Southern States hookworm work but has failed to find cases in
man. The eggs are not only larger than hookworm ones but show later stages of
segmentation.

Triodontophorus deminutus. — This is a small round worm with three forked teeth

HOOKWORMS

325

taking origin from the pharyngeal lobes. The collar-like mouth orifice is made up of
22 rounded plates just inside the round mouth opening. They are less than ^
inch long and have once been found in the intestinal canal.

(Esophagostoma brumpti.— Six young females were found in a cyst of the colon in
an African negro. They were about >£ inch (8 mm.) long. The anterior end pre-
sents an ovoid protuberance with a second cuticular inflation just below it. The
buccal capsule is very shallow and surrounded by about a dozen chitinous plates.
The mouth has six papillae.

This species has recently been reported by Thomas in a native of Brazil.

Physaloptera caucasica.— Mouth with two equal laterally placed lips, each hav-
ing three papillae and three teeth. The male has a lancet-shaped posterior extremity
and is about % inch long (14 mm. by 0.71 mm.). Female is about i inch long (27
mm.) with a rounded tail end. Found only once in the alimentary canal of a
native in the Caucasus. Leiper has recently reported a species P. mordens from
Uganda, one case.

THE HOOKWORMS OP MAN

The hookworm infections of man come almost entirely from two
parasites, Ancylostoma duodenale, the Old World species, and Necator
americanus, which is generally called the New World species from its
having first been reported from the U. S. by Stiles. Hookworms
belong to the class Nematoda and family Strongylidae.

Quite recently Lane has reported a new species, A. ceylanicum, as having been
obtained from three men in Bengal, after treatment. This species is the one that
infects the civet cat in Ceylon. So far as we know the other human species belong
solely to man.

The male hookworms are a little more than J£ inch (9 mm.)
long and the females a little more than J^ inch (13 mm.) in length.
The males can readily be distinguished by their posterior, umbrella-like
expansion or copulatory bursa. The tail of the female is pointed. The
vulva of A. duodenale is located in lower half of the ventral surface;
that of N. americanus in upper half. The large, oval mouth of the Old
World hookworm has four claw-like teeth on the ventral side of the buc-
cal cavity and two knob-like teeth on the dorsal aspect. It also has a
pair of ventral lancets below the four ventral teeth. One cannot make
out a dorso-median tooth. In N. americanus the buccal capsule is
round, smaller and the ventral teeth are replaced by chitinous plates.
Dorsally there are two similar but only slightly developed lips or plates.
A very prominent, conical dorso-median tooth projects into the buccal
cavity. Through it passes the duct of the dorsal cesophageal gland.
There are also four buccal lancets. The copulatory bursa of the Necator

326

THE ROUND WORMS

:left

americanus is also different, being terminally bipartite and deeply c
in the division of the dorsal ray, rather than tripartite and shallow, as
with A . duodenale. The anterior extremity of A ncylostoma bends in the
same direction as the general body curve while that of Necator hooks
back in an opposite direction to the body curve. In general, Ancylos-
toma is larger and thicker than Necator.

• . . . i . . . . I

SCALE:

FiG.76. — la, Copulatory bursa of Nectar americanus, showing the deep cleft
dividing the branches of the dorsal ray and the bipartite tips of the branches; also
showing the fusion of the spicules to terminate in a single barb. Scale ^0 mm-
ib, Branches of dorsal ray magnified. 2a, The buccal capsule of N. americanus.
2b, The same magnified. 3a. Copulatory of Ancylostoma duodenale, showing
shallow clefts between branches of the dorsal ray and the tridigitate terminations.
Spicules hair-like. 3b, The dorsal ray magnified. 4a, The buccal capsule of A.
duodenale, showing the much larger mouth opening and the prominent hook-like
ventral teeth. 4b, the same magnified. $a, Egg of N. americanus. sb, Egg of
A . duodenale. 6a, Rhabditif orm larva of Strongyloides as seen in fresh faeces. 6b,
Rhabditiform larva of hookworm in faeces eight to twelve hours after passage of
stool.

The name hookworm was given to these nematodes from the hook-like processes
of the ribs of the rays of the copulatory bursa. Dubini called the Old World para-
site Agchylostoma, properly Ancylostoma, on account of the four formidable hook- or
claw-like ventral teeth of the buccal capsule. (<ryx«^0<r, hook, and oro/xa, mouth.)

A. ceylanicum is somewhat smaller than A. duodenale and in the copulatory bursa
of the male we have a deeper cleft in the dorsal ray and two rather long tips to each

ANCYLOSTOMA AND STRONG YLO1DES 327

branch instead of the shallow cleft and three stumpy processes of the two branches as
in A. duodenale.

Goeze found a hookworm in a badger in 1782. He named the parasite Ascaris
criniformis. Froelich, in 1789, found hookworms in the fox and called them hook-
worms from the hook-like ribs of the copulatory bursa. He proposed the generic
name Uncinaria. Therefore Uncinaria belongs to the hookworms of the fox and
is not valid for any human species.

In 1838, Dubini found a hookworm as a human parasite. On account of the
four ventral teeth projecting from the mouth he gave it the name A gchylostoma or
correctly Anydostoma.

Bilharz and Griesinger noted the connection of the parasite with Egyptian
chlorosis, but it was not until the time of the St. Gothard tunnel (1880), that the
importance of the parasite was recognized. Grassi noted the diagnostic value of
the ova in faeces in 1878. In 1902, Stiles noted and described the hookworm found
in the United States as different and proposed the name Uncinaria americana, later
changed to Necator americanus. A. J. Smith had also recognized the morphological
differences.

Hookworms may be found in the small intestine (jejunum) of man
in enormous numbers. They either produce their effects by feeding on
the mucosa or by causing loss of blood.

Life History. — The delicate-shelled eggs pass out in the faeces, and
in one or two days a rhabditiform embryo (200 X 14 microns) is pro-
duced. The mouth cavity of the embryo is about as deep as the diame-
ter of the embryo at the posterior end of the mouth cavity; that of
Slrongyloides is only about one-half as deep as the diameter.

As a practical point, the anaerobic conditions in the intestines seem to prevent
development of the hookworm ova or at any rate the absence of the oxygen, so nec-
essary for the segmentations preliminary to the formation of the embryo, prevents it.
Therefore hookworm ova in freshly passed faeces never show other than commencing
segmentation while development of the larvae of Strongyloides takes place in the in-
testines, so that in freshly passed faeces we find, generally, actively moving larvae or
at least eggs containing fully developed embryos. Hookworm ova very rarely show
more than four segmentations or exceptionally eight in the freshly passed egg.

In the presence of oxygen these ova rapidly develop into larvae, particularly at a
temperature of about 27°C. Beyond 37°C. and below i4°C. development does not
seem to take place.

The rhabditiform larvae grow rapidly and by the third day are about
300 microns long and undergo a primary moulting. By the fifth day
the bulb-like swellings disappear and the larva becomes possessed of a
straight oesophagus, thereby becoming a strongyloid larva. It then
undergoes a second ecdysis or moulting, but instead of casting off this
old covering, it retains it as a protecting sheath. At this time it ceases
to take food but can move actively in its sheath so that it can crawl up

328 THE ROUND WORMS

blades of grass or vertical sides of mines. They can live in this stage
for months, when moisture and shade are present, but are rapidly
killed by drying.

This is the infecting stage in which the larvae bore their way into the
skin, which is the usual method of infection, or, occasionally, by enter-
ing the mouth on vegetables or otherwise.

Looss thought that they entered the skin by way of the hair follicles but the idea
now is that they can bore into any part of the skin. It only requires a few minutes
for the larvae to enter the skin. From the subcutaneous tissues they effect an en-
trance into lymphatics or veins, go to the right heart, thence to lungs. From the
alveolar capillaries they pass into the pulmonary alveoli, thence up the bronchi and
trachea, to pass out of the larynx and then down the oesophagus to the stomach.
The larva loses its protecting sheath in the stomach and in a few days develops a
provisional buccal capsule.

By the end of the second week, after another ecdysis, the larvae have grown to be
about 2 mm. long and 130 microns broad and in about four weeks become adults, usu-
ally in the jejunum, where, after fertilization of the females by the males, the giving off
of eggs begins. The adults attach themselves to the mucosa of the intestine, feeding
on the deeper structures of the mucosa, or on the tissues of the submucosa. Sambon
believes that the larvae can work their way into the jejunum without going there by
way of the trachea and oesophagus.

By providing an exit to the trachea, Fiilleborn demonstrated that in dogs, in-
fected with the dog hookworm, great numbers of larvae poured out of the trachea.
In other dogs he stitched the oesophagus to the skin and noted larvae coming out of
these openings. In these dogs, with the ordinary channel obstructed, infection did
occur with, however, only a few worms, thus showing the truth of Sambon's views
but at the same time demonstrating the unimportance of such a route of infection.

The mouth cavity of the embryo is about as deep as the diameter
of the embryo at the posterior end of the mouth cavity; that of Strongy-
loides is only about one-half as deep as the diameter. There is also a
globular expansion at the bottom of the mouth cavity with hook-
worm embryos while with Strongyloides ones the passage to the oesoph-
agus is funnel-shaped. Also the genital anlage of Strongyloides is much
larger than that of Ancylostoma.

A temperature of i°C. kills the eggs in twenty-four to forty-eight hours. After
moulting twice, it remains rather quiescent but still lying inside the discarded skin.
It reaches this stage in from four to fourteen days according to the temperature.

The soil in the area of the hookworm-egg-laden stool becomes infested with these
larvae which will even climb up blades of grass. It is for this reason that children
with their bare feet are so liable to infection.

Laboratory Diagnosis. — As a matter of fact the diagnosis is almost invariably
made by finding hookworm ova in the faeces. The eggs are oval and thin-shelled
with a wide, clear, glassy zone separating the more or less segmented, granular

HOOKWORM OVA IN F^CES

329

central portion from the shell. Formed stools are more satisfactory for examination
than the liquid ones resulting from a dose of salts. Put about 2 drops of water or
i% trikresol solution in the center of a glass slide and emulsify in it as much of the
faeces as is held by the spatulate end of a wooden toothpick. A small piece of wood
or a match stick will answer. These preparations can be readily examined without
a cover-glass, using a %-inch objective, with a i-inch ocular.

It is usually stated that about 500 worms must be present for several months
to produce symptoms. Grassi has thought that the presence of 150 eggs in o.oi
gm. faeces indicates the presence of 1000 worms, of which 25% would be males.

V)_

2

0

tt~

U-^

i-

0"

o-

ORAWN TO .C.ALC x IOOO

A.- Median focus BrSurf&ce focus

ArShowind f/
sides syif
metrically
convex

Oxyuris

vermicularis A.B.

DrAtypicoJ. unfertilized

e,§? (ftftcr Looss.lQQ^J

Strongylus
subtilis '

(fcf t«r Looss,

-."Without outer

t*H I •"* .!"'/( i*\ Tn^rMOiA TITIC« iBf sJ&

^^ Embryo

ry ,-, „ „ ^ in stool

^^/ An_y \fl^eri2to48

A0chylostonicx r AgchyibstomS

Trir*hi iri K ^f^S&T*3^*1'"2^ Jn'froshauxjl Abolt^«ti9o?S)r
trtcjP-- Strongyloides

(Origi

FIG. 77. — Nematode ova.

There may be as many as 4,000,000 eggs in a stool. Bass has proposed the fol-
lowing method for the examination of faeces for ova.

The faeces, which have been made fluid, should be centrifuged and the supernatant
fluid containing vegetable debris poured off. The sediment contains hookworm
eggs. Then pour on sediment a calcium chloride solution of sp. gr. 1.050. Again
centrifuge and decant. Next add calcium chloride solution of a sp. gr. of 1.250 and
centrifuge. This brings to the surface the hookworm eggs which may be pipetted
off. As a rule, the finding of hookworm eggs is very easy without such a technic.

In certain cases, where a microscope is not available, the diagnosis may be made
by finding the worms in the stool following a thymol treatment.

330 THE ROUND WORMS

The presence of eosinophilia is of great assistance in diagnosis but it should be
remembered that not rarely severe cases of the disease fail to show any excess of
eosinophiles.

Charcot Leyden crystals are often present in hookworm stools.

It has been claimed that where ordinary microscopical examination for ova will
show 40% of infections and methods involving concentration 55% that cultural
methods will show 99%. A convenient method of culturing is to make a pile of filter-
paper circles of 2 inches diameter and about Y± inch high and place in the center of a
4-inch Petri dish. Fill the dish with water about to the height of the filter-paper
and spread a thick layer of faeces on the top of the filter-paper island. The larvae
hatch out in about six days and swim out into the clear surrounding water. They
are best found by centrifuging the fluid containing them.

ASCARID.E

These have three papillae or lips around the oral cavity, one dorsal
and two ventral. The male has two equal-length spicules. An in-
termediary host is not needed in the life history of this family.

A

B

FIG. 78.— Anterior extremity of Ascaris lumbricoides; A, seen from front; B, seen
from dorsal surface. (Tyson after Railliet.)

Ascaris Lumbricoides. — The male round or eel worm is from 5 to
8 inches (18 cm.) long and the female from 7 to 15 inches (30 cm.) in
length. They are grayish to reddish in color and are from % to Y±
inch (5 mm.) in diameter.

It is probably the most common parasite of man, especially in
children and as it does not require an intermediate host infection takes
place through food or drink or by fingers of children who have been
playing where soil pollution exists.

The normal habitat is the upper part of the small intestine, hence the ease with
which they are vomited up. The three papillae-like lips with a constriction just
behind are easily studied with a hand glass. The very long, whitish, convoluted,
thread-like tubes of the uterus lead to the opening of the vulva anteriorly and ven-
trally. The male has two large lance-like spicules which project from a subterminal

THE PIN WORM 331

cloaca. The posterior extremity of the male is curved ventrally and has seven
pairs of postanal papillae.

The body of the worm is transversely striated and resembles the ordinary earth-
worm, but is more grayish than red. The ova are very characteristic with a rough
mammillated exterior. This at times is shelled off and we have a smooth egg which
may be mistaken for eggs of other parasites. The eggs leave the body in the fseces
and after a long time — a few weeks to several months, according to temperature — •
develop an embryo which remains in the shell until swallowed by man. It is stated
that they will remain alive for years. On being swallowed, the embryo leaves the
egg and we have males and females developing in the small intestine. In countries
where such parasites abound, as in Guam and the Philippines, the possibility of their
getting into the peritoneal cavity through operative measures on the intestine must
always be thought of.

Guiart considers it probable that Ascaris may suck blood, produce intestinal
ulcerations and bacterial infections, and perforate intestine. Their entrance into
bile ducts or into larynx (vomited) must be considered.

At autopsy they may be found perforating the appendix or even filling up the
pancreatic duct.

Some think that the symptoms of itching of nose and anus, vertigo, or convulsions
and anasrhia may be due to a toxin secreted by the worm.

Belascaris mystax. — This is a very common parasite of the dog and
cat, but is occasionally found in children. It is much smaller than the
A. lumbricoides— male is 2 to 3 inches (5 cm.) long, female 4 to 5 inches
do cm.) in length. The parasites are characterized by the presence
of wing-like projections from the anterior end farrow-like head). The
egg shells are quite thin.

Leiper has reported an infection with Toxascaris limbata in an
Egyptian. This is the smaller Ascaris of the dog.

Other Ascaridae reported from man are A. texana and A. maritime,
only one case each.

Oxyuris vermicularis. — The parasite is also known as the pin-worm
or seat-worm and is more frequent in children than in adults.

The male is about He inch (4 mm.) long and the female a little less than ^
inch (12 mm.) in length. The male has an incurved tail with a single spicule and the
female a long tapering tail. The vulva is in the upper third.

These worms have a clear slightly bulbous Turkish pipe mouth-piece-like projec-
tion surrounding the three-lipped anterior extremity. There is a well-marked bulb
oesophagus.

The eggs are thin-shelled plano-convex, and show a coiled-up embryo. After
ingestion of eggs, the adults develop in the small intestine where copulation takes
place; the males then die. The fertilized females go to the caecum and colon where
they remain until they reach maturity. At this time the females wander to the rec-
tum where they either expel their ova or themselves work their way out of the anus.
This usually occurs at night, and the scratching induced by the itching causes the

332

THE ROUND WORMS

wander

eggs to be widely spread about the region of the anus. The worms may also wandc
into the vagina, urethra, or under prepuce. It will be seen that as a result of the
scratching, the fingers become contaminated with ova which may be carried to the
mouth and so cause a fresh infection, no intermediate host being required. The
examination of the material under the finger nails of children harboring this parasite
may show eggs under the microscope. A knowledge of the life history— the early
location in the small intestine, and later on in the large — shows that treatment should
be dual in its direction — enemata for the gravid female in the rectum and santonin
and calomel for the young adults in the small intestine.

The diagnosis is preferably made by examining the stools for the
white, thread-like females which are expelled after a diagnostic dose of
calomel and salts, rather than by searching for the eggs.

These females, which are packed with embryo containing eggs, may be seen
wriggling on the surface of the freshly passed faeces. In handling these worms
one should be careful as they are apt to cause infection should the eggs get on the
fingers.

ACANTHOCEPHALI

These are called thorn-headed worms on account of a proboscis which projects
anteriorly like a little peg.

There are several rows of hooks surrounding this projection which are directed
backward to enable the parasite to attach itself to the intestinal wall. The worm
absorbs nourishment through the general body wall, there being no alimentary
canal or mouth. These worms are common in hogs. The three-shelled eggs are very
striking and the intermediate stage is in June bugs.

The Echinorhynchus or Gigantorhynchus gigas. — This parasite is about 6 inches
(15 cm.) long for the male and 10 to 12 inches (25 cm.) for the female. It has
transverse rings and resembles Ascaris but is more white in color. It is said to be not
uncommon in southern Russia.

The Echinorhynchus or Gigantorhynchus moniliformis might be contracted
by persons eating death-watch beetles as is sometimes done for the improvement of
the complexion.

HIRUDINEI (LEECHES)

Hirudo medicinalis. — This is the leech used medicinally for the abstraction of
blood. They have a secretion which prevents coagulation of the blood so that when
they are removed the wound still continues to bleed.

Limnatis nilotica. — This species has been found in many parts of northern Africa
and, gaining access to the stomach through drinking water, it wanders to the phar-
ynx, nares, and even trachea. Manson refers to a case of obstinate epistaxis and
headache caused by a leech in the nostril.

This leech is about 4 inches long (8 to 10 cm.) and about % inch (i.a cm.) broad.
The dorsal surface is greenish brown with narrow orange-brown borders. The young
leeches are only about )£ inch (3 mm.) long and taken in with the drinking water

LEECHES 333

may attach themselves to the surface of some mucous membrane and after some
weeks reach adult size.

Hsemadipsa ceylonica.— These are land leeches found in India, Philippines,
Australia, and South America. They are only about i inch (25 mm.) long and are
slender. They leave the damp earth to climb shrubs and from there to drop on
animals or man passing through the forest. The bites are painless, but may be
followed by ulcers. They may get into the nostrils.

They will even penetrate thick clothing in order to reach the skin.

CHAPTER XIX

Order

Acarina

THE ARACHNOIDS

CLASSIFICATION or THE ARACHNOIDEA
Family Subfamily Genus

Trombidiidae
Gamasidae

Tyroglyphidae
Sarcoptidae
Demodicidae
Tarsonemidae

Argasinae

Species

Ixodidae

Ixodinae

Trombidium

T. holosericeum

Dermanyssus

D. gallinae

Tyroglyphus

f T. farinae
\ T. longior

Sarcoptes

S. scabiei

Demodex

D. folliculorum

Pediculoides

P. ventricosus

(Argas

! A. persicus
\ A. miniatus

Ornithodoros

O. savignyi

Ixodes

I. ricinus

Hyalomma

H. aegyptium

Rhipicephalus

R. bursa

Dermacentor

f D. reticulatus
\ D. andersoni

Margaropus

M. annulatus

Amblyomma

A. hebraeum

Haemaphysalis

H. leachi

Lingua tula

L. rhinaria

Porocephalus

P. constrictus

Pentastomida

The class Arachnoidea and the class Insecta belong to the phylum
Arthropoda. This phylum contains a greater number of species than
does any other phylum.

While the lobsters, crabs and water fleas, which belong to the class Crustacea, are
important zoologically, they are of very slight importance medically. Besides the
Crustacea we have the thousand-legged worms or Myriapoda.

The different classes of Arthropoda resemble the segmented worms
but have as distinction the possession of jointed appendages which
proceed from the somites in pairs. Some of the pairs of limbs are for
locomotion; at times, certain ones may be specialized for food taking.

The somites or divisions of the body have a chitinous exoskeleton.

334

MITES 335

Respiration takes place through the medium of gills in the Crustacea
and by tracheal tubes in the Myriapoda, Arachnoidea, and Insecta.

The Arachnoidea have no antennae while the Myriapoda and Insecta have a
single pair of antennae, the former having numerous pairs of legs or jointed appen-
dages while the latter have only three pairs of legs. The Arthropoda have seg-
mented bodies, but they differ from the worms in having jointed appendages for the
purpose of .taking in food and moving from place to place. They also have an
exoskeleton which is more or less unyielding from the deposit of chitin in the cuticle.
This cuticle is not a true skin but only a secretion of the epidermis.

Within this external skeleton we have a dorsal digestive system and
a ventral nervous system.

THE ARACHNOIDEA

The Arachnoidea differ from the Insecta in having the head and
thorax fused together. They also have four pairs of ambulatory appen-
dages, while the insects only have three pairs. The Arachnoidea never
have compound eyes — these when present being simple. Of the two
orders of Arachnoidea of interest medically the Acarina is far more
important than the Linguatulida.

ACARINA

Of the acarines we are chiefly interested in the mites and the ticks.
The acarines do not show any separation of the abdomen from the
cephalo-thorax. A hexapod larva develops from the egg; this is suc-
ceeded by an octopod nymph which differs from the adult in not having
sexual organs.

In addition to the four pairs of legs in the fully developed acarine there are two other
paired appendages, the chelicerae, in front of the mouth, and the pedipalps on
either side of the mouth.

Trombidiidae

These generally have a soft, more or less hairy integument and are often brightly
colored. The two eyes are often pedunculated and the chelicerse are lancet-shaped
and the palps project beyond the rostrum as claw-like appendages. A tip-like
appendage on the apical segment of the palps is characteristic. A very common
and annoying member of this family is the hexapod larva of the Trombidium
holosericeum. It is usually designated Leptus autumnalis. Popularly it is termed
"harvest mite," "red bug" or "jigger." They are found in the fields in the
autumn and attack both man and animals. The condition (itching and redness)

336

THE ARACHNOIDS

produced is at times called autumnal erythema. There is a Trombidium in Mexico
which has a predilection for the skin of the eyelids, prepuce, and navel. The Kedani
mite, an orange-red larval mite about 250 by 125 microns is believed by the Japanese
authorities to bring about infection with Japanese river fever or Tsutsugamushi,
as the result of transmitting either a bacterium or protozoon by its bite. The
disease somewhat resembles typhus, although an eschar at the site of the bite and
lymphatic involvement is present.

Gamasidae

Of the Gamasidae, which generally have a hard leathery body and styliform
piercing chelicerae, delicate five jointed palps and styliform hypostome, only the
Dermanyssus gallina is of interest. This mite infests chicken-houses and sucks the

FIG. 79. — Arachnoidea exclusive of ticks. (ia) Sarcoptes scabiei, female; .(ib) S.-
scabiei, male; (2) Demodex foUiculorum; (3) Trombidium akamushi, hexapod larva
(Kedani mite); (4) Trombidium holosericeum larva (Leptus); (5) Dermanyssus
gallina; (6) Tyroglyphus longior; (7 a) Pediculoides ventricosus, male; (76) P. ventri-
cosus, young male; (7^) P. ventricosus impregnated female; (8) Porocephalus armll-
latus; (90) Linguatula serrata, female; (96) L. serrata, larva.

blood of the inmates. They will also attack man. Poultrymen may be troubled
with a sort of eczema on the backs of the hands and forearms, similar to scabies,
resulting from bites by these mites. They measure 350 X 650^1. They have no
eyes.

Tyroglyphidae

Mites of this family live on cheese, flour, dried fruits, etc. They are small,
without eyes, and have a smooth skin and a cone-like appearance of the mouth

THE ITCH MITE 337

parts which are largely formed by the chelate cheliceras. They are chiefly of
importance because of their being occasionally found in urine, faces, etc., and being
striking objects, the question of pathogenicity arises. The T. longior has been
associated with intestinal trouble (probably a coincidence, patient having eaten
cheese containing these mites).

Glyciphagi are found in sugar and are the cause of what is known as "grocers'
itch." Rhizoglyphus parasiticus is reported to be the cause of an itch-like affection
of the feet of coolies on tea plantations. To distinguish: the dorsum of Glyciphagus
is hairy or plumose; Tyroglyphus has both claws and suckers on tarsi, while Rhizo-
glyphtis has only claws.

Sarcoptidae

These are small eyeless mites with a transversely striated cuticle. They live
on the epidermis of man and various animals. The rostrum is chiefly made up of
chelate chelicerae with quite short three jointed, rather adherent palpi. It is the
female that makes the tunnels in the skin between the fingers, on penis, flexor surface
of forearm, etc. The male dies off after copulation. The female passes through four
stages: i. larva; 2. nymph; resembles adult, but has no sexual organs; 3. the pubes-
cent female; 4. the egg-bearing female. A pair of itch mites may produce 1,500,000
descendants in three months. Transference of eggs, larvae or pubescent females
does not seem to transmit scabies. It is the egg-laden female only. The human
itch mite, Sar copies scabiei, is an oval mite, the male is 250 X 150/^5 the female is
about 400 X SOOJL*. Besides the difference in size, the male may be distinguished
from the female by the fact that the third and fourth pairs of legs in the female have
bristles, but in the male, the fourth pair has suckers. The tunnels made by the
female have the egg-bearing female at the blind end; scattered all along are fasces,
eggs, larvae; the eggs being next the mother and the more mature young at the en-
trance to the gallery. A diagnosis can be made from the finding of either eggs or
larvae. The eggs are 140;* long and hatch out in four to five days. A female becomes
mature in about two weeks.

In treating itch with sulphur preparations the adult females and immature itch
mites are killed; the eggs, however, are not affected. Hence a second treatment
about ten days after the first is necessary to kill the young mites, which have devel-
oped subsequent to the first treatment. Different animals have different species of
itch mites.

Demodicid® (Hair Follicle Mites)

Demodex folliculorum. — This is a vermiform acarine about 400 JU long; the eggs
are about 75/z long; they chiefly live in the sebaceous glands of nose and forehead.

Tarsonemidae

This acarine family shows a complete sexual dimorphism. The Pediculoides
ventricosus is oval and about 125 X 75M for the male which has claws at the extremi-
ties of the anterior and posterior pairs of legs; the two other pairs have booklets and
a sucking disc. The female is about twice as long but of the same breadth as the
male, and has claws only on the anterior legs.

338 THE ARACHNOIDS

The chelicerae are needle like with inconspicuous palps and the front and rear
pairs of legs are widely separated. The gravid female is like a ball and is about
looofA in diameter.

They live on wheat and may be found in wheat straw, which, if handled, may be
followed by a severe skin eruption with an irregular fever.

Ixodidae

This family of the Arachnoidea is one of great medical interest and
of growing importance. It has recently been proposed to raise the
ticks to a superfamily, Ixodoidea and to divide it into the families
Argasidae and Ixodidae.

While only proven the intermediary hosts in the case of the organism of African
tick fever and the recently discovered cause of spotted fever of the Rocky Moun-
tains, there is considerable speculation as to the possibility of blackwater fever being
due to a Babesia (Piro plasma}. Piroplasmata of animals seem to be invariably
transmitted by ticks.

Very important diseases due to these small pear-shaped organisms within red
cells are known for various animals, the best known being that of cattle in Texas
and known as Texas fever. Other piroplasmata diseases are Rhodesian fever
(cattle), heart water (sheep), and malignant jaundice of dogs. In these diseases
there are pathological features which resemble blackwater fever of man.

It is of interest to note that it was with the transmission of Texas
fever through an intermediate host (the tick) that Smith and Kilborne
(1889-1893) established the zoological principle of transmission of
disease through arthropod intermediary hosts. This led up to the
work on malaria, yellow fever, etc.

Ticks differ from insects in having four pairs of legs, only two pairs of mouth
parts, and no antennae. They differ from other acarines in having a median probe-
shaped puncturing organ, the hypostome, which is beset with numerous teeth
projecting backward, and in possessing stigmal plates. The head, or capitulum,
or rostrum, is the part which projects anteriorly from the body. This carries the
piercing parts which are the single hypostome or dart and a pair of piercing chitinous
structures, the chelicerae which lie above the hypostome. As a sheath for these
delicate biting parts we have a segmented pair of palpi or pedipalps. The mouth
is a slit between the chelicerae and hypostome.

Two depressed pitted areas on the dorsal surface of the capitulum in the adult
female are known as porose areas. Very important structures are the stigmal
plates. These are striking mosaic-like areas which are located just posterior to
each hind leg in the Ixodinae and between the third and fourth legs in the Argasinae.
As the greatest confusion exists as to the classification of ticks, Dr. Charles W.
Stiles has now in hand a system of classifying ticks according to the appearance
of these plates as seen under the high power of a microscope. There is great varia-

TICKS 339

tion in the outline and general picture of these stigmal plates in the different species.
The stigmal orifice, the opening of the tracheal system, is in the center. The
Ixodinae have a scutum- or shield-like chitinous structure on the dorsal surface. It
covers almost the entire back of the tick in the male and only a small portion ante-
riorly in the female. The genital opening is toward the anterior part of the ventral
surface. The anus, with anterior or posterior anal grooves, is near the posterior
third of the venter. The legs have six segments, the coxa being flattened out on the
surface of the body and the terminal tarsus.ending with a pair of hooks and at times
with a pulvillus. The nymph has stigmal plates but has no genital opening while
the larva has neither genital apertures nor stigmal orifice.

Life History of Ticks. — This varies greatly according to the sub-
family, genus, and species. The female Ornithodoros samgnyi lays
about 140 eggs. The larva does not leave the egg, but moults inside,
and finally emerges as an eight-legged nymph. It lives in the dust in
the cracks of the native huts and comes out at night to feed on the sleep-
ing natives. As the possibilities for destruction are not so great as with
many Ixodinae the necessity for thousands of eggs is not imperative for
the continuation of the species as with the Ixodinae. With some of
the Ixodinae the females lay from 5000 to 20,000 eggs during several
days or weeks and then die. The eggs are preferably deposited near
grass. The egg stage lasts from two to six months, when the six-legged
larva ("seed tick") emerges. It crawls up a blade of grass and gets on a
passing animal. After feeding, or at times without taking nourishment,
the larva drops to the ground, and changes to the pupal stage which has
four pairs of legs. The pupa crawls up a blade of grass and gets on a
passing animal (the second host). Feeding, it falls to the ground where
it remains eight to ten weeks. It moults and develops into an adult
tick. These males and females gain access to a third animal host—
the males fecundate the females, after which the female gorges herself
with blood; afterward dropping off the animal and laying eggs. With
some ticks fewer hosts suffice.

Cleland has noted reports of serious symptoms, chiefly cardiac and visual, from
the bite of ticks in Australia (Ixodes holocydus). This is exceptional, however, as
the symptoms following the bites of such ticks are only those of skin irritation.

Classification of Ixodidae

Subfamily Argasinse. — Head concealed by body when viewed
dorsally. No scutum. Stigmal plates between third and fourth legs.
Adults have no suckers (pulvillus) beneath claws. Slight sexual
dimorphism. Anus near middle of venter. Skin rough.

340

THE ARACHNOIDS

Genus Argas. — Body narrow in front. Margins thin and acute. No eyes.
The A. persicus (Miana bug) of Persia has been supposed to be concerned in the
transmission of a serious disease. Rostrum some distance from anterior margin.
It is also called the fowl tick and transmits fowl spirillosis.

Genus Ornithodoros. — Margins of body rounded. Skin has many irregular
tubercles. Rostrum even with anterior margin so that ends of palpi slightly project.
It is the intermediate host of Spirochata duttoni. (South African tick fever.)

0. moubata is very common in Africa living in cracks in mud floors and bites
severely the sleeping natives. The larva makes its first moult inside the egg so
that it shows four pairs of legs when it emerges. Christy thinks it may transmit
Filaria perstans.

Tick Fever. — With tick fever the epidemiology rests upon the
life history of the tick 0. moubata. This tick infests the rest houses

FIG. 80. — Ornithodoros moubata. (Murray from Doflein.)

along the route of travel, hiding in the crevices of floors and walls during
the day and coming out at night to bite the sleeping inmates. The
feeding occupies a long time, more than an hour. Both sexes bite man.
The female lays about 100 eggs, from which a nymph emerges in about
twenty days. The larval stage takes place in the egg. Shortly after
emerging the nymphs suck blood. An important fact is that the female
transmits the spirochaete to its ova, so that the nymphs may transmit
the disease.

Natives seem to sutler severely from tick fever in childhood but in adult life
possess a sufficient degree of immunity so that the disease shows itself in a very
mild form in those harboring spiroch<etes. Ticks can be infected by these carriers.
In some of the rest houses 50% of the ticks may be infected. While the tick does not

TICK FEVER

341

00

342 THE ARACHNOIDS

tend to leave its habitation it may be transported in the bundles of native porters.
The transmitting agent of the north African relapsing fever and probably of the
Indian type is the louse. The body louse deposits about 75 eggs in the clothes of the
host, which hatch out in about four days and become adults in about two weeks. The
head louse deposits its eggs or nits on the hair of the host's head. (See Pediculus.)
O. savignyi has two pairs of eyes near base of mouth parts.

Subfamily Ixodinae. — Mouth parts project in front* of body when
viewed dorsally. Scutum present. Stigmal plates posterior to fourth
pair of legs. Adults have suckers beneath claws. Skin finely striated.

Anus behind middle of venter.

Sexual dimorphism marked. Male has well- developed scutum;
female has porose areas.

Section Ixodae. — Transverse recurved preanal groove in female. Male has ventral
surface covered with chitinous plates. No eyes. Genus Ixodes.

Ixodes has long rostrum with slender palpi — palpi narrow at base, leaving gap
between them and hypostome.

Section Rhipicephalus. — No preanal, but postanal groove in female. Ventral
surface of male without adanal plates in Dermacentor, Hcemaphysalis^ Aponomma
and Amblyomma, but with one or two pairs in Hyalomma, Rhipicephalus and
Margaropus.

In the genera Hyalomma, Aponomma and Amblyomma the palpi are long and
slender and of about uniform width of segments.

In Hyalomma the segments of palpi are of about equal length. In Aponomma
and Amblyomma the second palpal segment is much longer than the others. Ambly-
omma differs from Aponomma in being very ornate and in having eyes.

In the genera Hcemaphysalis, Dermacentor, Rhipicephalus, and Margaropus the
palpi are short.

Hcemaphysalis has very broad rostrum, triangular palpi, and no eyes. Derma-
centor has a square rostrum with short thick palpi, the second and third joints being
as broad as long. Dermacentor andersoni transmits spotted fever of the Rocky
Mountains — not D. reticulatus. The most important characteristic of the genus
Dermacentor is the large size of the coxae of the fourth pair of legs.

Rhipicephalus has palpi without transverse ridges and comma-shaped stigmal
plates. The stigmal plates of Margaropus are nearly circular and the palpi have
acute transverse ridges externally. Margaropus annulatus transmits Texas fever
of cattle. This tick is also called 'Boophilus bovis or B. annulatus. Some authors
term it Rhipicephalus annulatus. Larvae developing from eggs of female ticks
which have fed on cattle infected with Texas fever transmit the disease which is due
to a protozoon Babesia bigemina.

PENSTASTOMIDA (TONGUE WORMS)

These are vermiform acarines more or less distinctly annulated. They have
retractile hooks at either side of the elliptical mouth.

TONGUE WORMS 343

If the hooks are to be considered not as degenerated legs but antennse and palpi,
then there is no vestige of legs in the adult. The sexes are separate.

Linguatula rhinaria. — This has been observed in man both in larval and adult
stages.

The male is white and about Y± inch long while the female is about 4 inches
long, tadpole shape, yellowish in color, and has about 90 segments, lives in the
nasal cavity and frontal sinus of dogs, rarely in horses and sheep, and very rarely
in man.

The female lays embryo-containing eggs which, gaining freedom through the
nasal mucus, are swallowed by various animals. A larva develops which bores its
way through the gut and encysts in the liver or mesenteric glands. After several
moultings, they work their way again to the intestines and so get out of the body
of their host; or they may wander to lungs and trachea and either escape or take up
their position in the nostrils to become adults and produce eggs. Consequently,
one animal may act as intermediate and definitive host or these cycles may take
place in distinct animal hosts.

The larval form (^ inch) is far more common in man than the adult. Symptoms
are referred to liver in both larval and adult stage, and epistaxis and nasal symptoms
for adult stage only.

Porocephalus constrictus. — The adult form P. moniliformis lives in the lungs of
snakes and the eggs are probably ingested by drinking water. These eggs develop
into a curled-up, ringed larva, about ^ inch long with 23 rings, which is
encysted especially in the liver or lungs. These escape and are swallowed by the
snakes, their definitive hosts.

While in the liver or lungs of man the patient may have signs of bronchitis, hep-
atitis or peritonitis. Cases usually only discovered at postmortem. Parasites,
however, might possibly be found in sputum or faeces.

CHAPTER XX
THE INSECTS

CLASSIFICATION OF THE CLASS INSECTA

Order Family

Subfamily Genus

Species

Siphunculata

Pediculidae

1 Pediculus

P. capitis
P. vestimenti

[ Phthirius

P. pubis

Rhynchota

Acanthiidae

Acanthia

A. lectularia

(Hemiptera)

Reduviidae

Conorhinus <

C. megistus
C. sanguisuga

Pulex

P. irritans

Xenopsylla

X. cheopis

Pulicinae -

Ceratophyllus

C. fascia tus

Siphonaptera •

Pulicidse

Ctenocephalus

C. serraticeps

Ctenopsylla

C. musculi

Sarcopsyl- Sarcopsylla

S. penetrans

linae

Simulidae

Shnulium

S. reptans

(buffalo gnats)

Psychodidae

Phlebotomus

P. papatasii

(moth midges'

Chironomidae

Ceratopogon

C. pulicaris

(midges)
Culicidse

'Culicin* /Stegomyia
\ Culex

S. calopus
C. fatigans

Anophe- Anopheles

A. maculipennis

linae

Tabanus

T. bovinus

Tabanidae

Haematopota

H. pluvialis

(horseflies)

Pangonia

P. beckeri

Chrysops

C. dispar

Diptera

Glossina <

G. palpalis

1

G. morsitans

Stomoxys

S. calcitrans

Muscidas

Musca

M. domestica

Auchmeromyia

A. luteola

Calliphora

C. vomitoria

Lucilia

L. caesar

Chrysomia

C. macellaria

(screw-worm)

Sarcophagidae

(Sarcophaga
Ochromyia

S. carnaria
O. anthropo-

haga

(Estridae

{Dermatobia
Hypoderma

D. cyaniventris
H. diana

344

LICE 345

INSECTA

The class Insecta has one pair of antennae, three pairs of mouth parts
(the fused labium being considered as one pair), and three pairs of legs.
They have three divisions of the body — head, thorax, and abdomen.

The head carries the antennae and mouth parts; the thorax, which is divided
into the pro meso and meta thorax, carries upon the ventral surface of each thoracic
segment a pair of legs and on the dorsal surfaces of the two posterior segments a
pair of wings. The abdomen does not support appendages. The air is supplied
by means of tracheae — branching breathing tubes which have external openings or
stigmata. The tracheae are stiffened by spiral chitinous bands. The Malpighian
tubules are excretory organs of the alimentary system and excrete nitrogenous
waste material. Insects have two pairs of wings, the second pair of which is fre-
quently rudimentary and shows simply as knob-like projections. These are termed
halteres or balancers. In some insects both pairs of wings are rudimentary, as in
Siphonaptera.

Where insects show metamorphosis we have voracious worm-like larvae coming
out of eggs; these larvse are succeeded by a quiescent no nf ceding encased pupa
which finally develops into an imago or fully developed insect. An insect which does
not present this developmental cycle shows incomplete metamorphosis. Of the
class Insecta only the Siphunculata, Rhynchota, Siphonaptera, and Diptera are of
special importance.

SIPHUNCULATA (ANOPLURA)

These are small dorso-ventrally flattened wingless insects not showing
metamorphosis.

The Pediculidae

In this family there are no wings and there is no metamorphosis.
The acorn-shaped eggs (nits) are deposited on hairs of the host.

Pediculus capitis. — The female is about % inch (3 mm.) long; the male
smaller. They vary in color according to the color of the hair of the host. The eggs
are deposited on the hairs of the head in number of 60 which hatch out in about six
days. The thorax is as broad as the abdomen. The male louse is rounded off
posteriorly and shows a dorsal aperture for a pointed penis, while the female is
recognized by a deep notch at the apex of the last abdominal segment. There seems
to be a marked preference exhibited by lice for their own peculiar racial host. It has
been suggested that this might account for certain peculiarities in infection where
different races were living together and under similar conditions as to food and
environment, and yet only one race contracts the disease (beriberi). The head louse
has been found to harbor leprosy bacilli when living on a leper.

Pediculus vestimenti. — This louse lives about the neck and trunk and deposits
its eggs in the clothing. They number about 75 and hatch out in three or four days
and become mature in about two weeks. Unlike the fleas there is no grub stage.

346

THE INSECTS

It is almost twice the size of the P. capitis and the abdominal seg-
ment is broader than the thorax. The abdomen is less markedly
festooned than that of P. capitis; is less hairy and contains 8 segments
as against 7 for P. capitis.

It has recently been shown to transmit typhus fever and more recently Nicolle
has demonstrated it as a carrier of relapsing fever, the spirochaetes being introduced
by the material from the crushed louse being rubbed into the wound by the scratch-
ing of the victim (just as with the flea in plague) and not by the bite itself. The
dog louse as well as the dog flea serves as an intermediary host for Dipylidium.

FIG. 82. — Siphunculata and Rhynchota. i. Pediculus capitis. 2, Pediculus
vestimenti. 2 a. Protruded rostrum of Pediculus. 3. Phthirius pubis. 4. Acanthia
lectularia. 5. A. rotundata. 6. Conorhinus megistus.

Phthirius pubis. — This louse is popularly known as the crab louse. The female is
little more than %5 inch in length, and the male a trifle less. They are almost
square. The second and third pair of legs are supplied with formidable hooks.
They have a preference for the white race and live about the pubic region. The
female lays about a dozen eggs, which hatch out in about a week.

RHYNCHOTA

The Rhynchota are insects possessing a sucking beak in which the
lower lip forms a long thin tube or rostrum which can be bent under the

BEDBUGS

347

head or thorax. Inside this tube are biting parts— mandibles and max-
illae. The metamorphosis in this order is not marked.

They have no palpi. The lower lip or labium or beak has its
edges curved to form the tube and it is only covered by the labrum at
its base. With the Diptera the labrum goes into the formation of the
sucking tube. The mandibles and maxillae are bristle-like structures
serrated at the tip. The mandibles are grooved internally and form
when apposed a tube for blood.

The Acanthiidae

These have a flattened body, a three-jointed rostrum, and four-
jointed antennae. Their wings are atrophied.

FIG. 83. — Fleas, bedbugs and ticks. A, Lcemopsylla cheopis; B, P. irritans;
C, Ctenopsylla musculi; D, bedbug; E, cross section of rostrum of Ornithodorus;
F. longitudinal section of Ornithodorus.

Acanthia lectularia (Cimex lectularius). — This is the cosmopolitan bedbug.
It measures about K by M inch (5 by 3 mm.). It is of a brownish-red color.
The most conspicuous feature of the bedbug is the long proboscis continuous with
the dorsal integument of the head and tucked under the ventral surface. There are
two prominent eyes and two four-jointed antennae. There are eight abdominal
segments. The bedbug lives in cracks and crevices, especially about beds. It is
said they can migrate from house to house. At any rate, they are frequently trans-

348 THE INSECTS

The

ferred with wash clothes. They have a penetrating odor when crushed. The
female deposits about 50 eggs at a time in cracks and in ten days 'they hatch out
into larvae which pass insensibly into adults by a series of five moultings; this deposit-
ing of eggs occurs about four times a year.

The bedbug is very probably the intermediate host in kala-azar and it has been
incriminated in connection with typhus fever and relapsing fever. It can also
transmit plague.

A. rotundata. — In India the A. rotundata is the one encountered. It is of a dark
mahogany color, has a smaller head, narrower abdomen, thick rounded prothoracic
borders and is more densely covered with hairs than A. leclularia. The prothorax of
A . leclularia is flattened at the side.

Reduviidae

These bugs have a long narrow head and a distinct neck. The
antennae are long and slender. The antennae in the genus Conorhinus
are inserted about midway between the eyes and point of the head.

Conorhinus sanguisuga. — This is known as the Texas or Mexican bedbug, and
was formerly the foe of the common bedbug, but having gotten a taste for human
blood through the Cimex or Acanthia, it now prefers man. It is extending toward
the North. It has wings. The bites are much more severe than those of the
common bedbug. It is of a dark brown color, nearly an inch in length, with a long,
flat, narrow head and a short thick rostrum. They can run as well as fly. They
bite at night.

Conorhinus megistus. — This is called "Barbeiro" in Brazil on account of its
preference for biting the face. The Schizotrypanum cruzi undergoes a develop-
mental cycle in this bug which transmits the disease.

SlPHONAPTERA

These are laterally flattened, markedly chitinized, wingless insects
which undergo a complete metamorphosis.

Pulicidse

This family is divided into two subfamilies — the Pulicinae and the
Sarcopsyllinae. In the former the female remains practically un-
changed with freedom of movement after fecundation, while in the
latter the abdomen becomes enormously distended with eggs, and the
female remains fixed in the burrow which she has made under the skin.

Pulcinse. — Formerly, with the exception of infection with Dipylidium caninum,
the fleas were only under suspicion as carriers of disease; ideas having been enter-
tained as to their being possible transmitters of relapsing fever, typhus fever and
kala-azar. Trypanosoma lewisi is transmitted by fleas, either Pulex irritans or
C. cams. The trypanosome undergoes development in the flea and the infecting

FLEAS 349

material is in the fasces of the flea and transmission occurs by the licking on the part
of the rat of faeces from an infected flea. The infection has no connection with the
puncture wound of the flea as is the case with plague. As a result of the convincing
experiments of the Indian Plague Commission, their role in the transmission of
plague has been absolutely established. It is by the bite of the Xenopsylla cheopis
that plague is chiefly transmitted from rat to rat, and in bubonic and septicaemic
plague it is apparently the intermediary in human infection.

The average capacity of a flea's stomach is about 0.5 cu. mm. so that with a rat
dying with septicaemic plague and with possibly 100,000,000 bacilli to i c.c. of
blood the flea would take in about 5000 bacilli. Furthermore these multiply in
the alimentary canal so that the digested blood teems with bacilli when reaching
the anus of the flea. The plague bacilli are passed out with the fasces and these
being rubbed into the puncture of the flea bite^ bring about infection. Regurgitation
as result of obstruction by masses of plague bacilli in the oesophagus causes in-
jection of plague bacilli into rat or man in the act of biting. This is more important
than the faeces inoculation method. The puncturing apparatus of the flea consists
of a pointed epipharynx and two distally serrated mandibles. These chitinous
biting parts are contained in the labium which divides distally into two labial palps.
The maxillae are conspicuous triangular structures and, projecting farthest anteriorly,
are the conspicuous four-jointed maxillary palps, often mistaken for antennae.
By the apposition of the internally grooved mandibles to the epipharynx a tube is
formed through which the blood is sucked up. The antennas are inconspicuous and
are in close apposition to the sides of the head, behind the eyes, and can only be
well made out with a lens. Fleas have three pairs of legs, and the male can be
distinguished from the female by its smaller size and the conspicuous coiled-up
penis within the abdomen. The female has a conspicuous gourd-like spermatheca
which varies in shape in different species. A very prominent structure is a pitted
plate in the ninth abdominal segment (pygidium). Of importance in classification
are prominent bristles originating from the seventh abdominal segments and
projecting over the pygidium. These bristles vary in number and are known
as antepygidial bristles.

The body of the flea is flattened laterally. They may or may not have eyes, and
certain conspicuous structures called combs are of importance in classification. In
the metamorphosis of the flea the eggs are hatched out in dust of crevices, etc., into
bristled larvse in about one week. The larva forms a cocoon and develops into a
nymph which has three pairs of legs. The ftymphs emerge from the cocoon as adult
fleas in about three weeks after the larva forms it.

KEY TO THE FLEAS
A. With combs,
i. Eyes present.

(a) Combs along inferior border of head and on prothorax.
Ctenocephalus serraticeps.

(b) Combs only on prothorax. Ceratophyllus fasciatus (with
only one antepygidial bristle on each side).
Hoplopsyllus anomalus (with many antepygidial bristles).

350

THE INSECTS

2. Eyes absent.

(a) Collar of combs on prothorax and four short ones along

inferior border of head. Ctenopsylla musculi.
B. Without combs.

(a) Ocular bristle arises near upper anterior margin of eye. A
line between this and the oral bristle approximately ver-
tical. Two bristles posterior to antennae. Xenopsylla
cheopis. Loemopsylla cheopis. Formerly Pulex cheopis.

-ZArery-

FIG. 84. — i and 2, male and female Xenopsylla cheopis. 3, Head of Ceratophyllus.
4 and 5, male and egg distended female of Sarcopsylla penetrans.

(b) Ocular bristle arises near lower anterior margin of eye. A
line between this and the oral bristle approximately hori-
zontal. One bristle posterior to antennae. Pulex irritans.

The common human flea of Europe is the Pulex irritans; that of the United States
the Ctenocephalus serraticeps or dog flea. The flea that is prominently implicated
with plague is the Xenopsylla cheopis, on account of its being the common rat flea
of India, where it has been much studied. It resembles P. irritans t but is more yellow
than brown in color. It also has a greater number of bristles on the head. The
ocular bristle runs above and in front of the eye; that of P. irritans below. It is
principally the flea of Mus decumanus (M. norvegicus), the sewer rat; but the house
rat, M . rattus, becomes infected from coming in contact with the sewer rat in the

THE CHIGOE 351

basement. In the U. S. the ground squirrel, Citellus beechyi acts as a reservoir of
plague and has as its flea Hoplopsyllus anomalus.

Ceratophyllus fasciatus is the common rat flea of Europe and the U. S. In the
tropics X. cheopis is the common rat flea (98% in India). Ctenocephalus serrati-
ceps, Ctenopsylla musculi and Pulex irritans have also been frequently found on
both Mus norvegicus and M. rattus. To distinguish M . norvegicus from M. rattus
we have in the former (i) ears which barely reach the eyes when laid forward and (2)
tail rather shorter than length of head and body together (only 89% of length of head
and body together). With M. rattus the tail is longer than the head and body
together (25% longer) and the extended ear covers or reaches beyond the middle of
the eye. M. rattus has a sharper nose, longer and more delicate tail and thinner ears
than M. norvegicus (formerly M. decumanus).

M. alexandrinus is a variety of M. rattus. Rats and mice belong to the family
Muridae and the common mouse is M. musculus. They belong to the order of
Rodentia of the class Mammalia.

Sarcopsyllinae

Belonging to the subfamily Sarcopsyllinae, the Sarcopsylla penetrans (Derma-
tophilus penetrans} is of great importance in tropical countries. It is known as
the chigoe, nigua, or jigger. The male and virgin female are unimportant as they
do not penetrate the skin but act as ordinary fleas. The female, which when un-
impregnated is only about ^4 inch long, when impregnated bores its way into
the skin of man, especially about the toes, soles of the feet or finger-nails, and in the
chosen site develops enormously, becoming as large as a small pea. This enlarge-
ment takes place in the second and third abdominal segments and is packed with eggs
measuring about 400 microns long and numbering about 100. A small black spot in
the center of a tense rather pale area is characteristic. The metamorphosis is
similar to that of the flea. Sarcopsylla can be differentiated from the flea by the pro-
portionately larger head to the body, and especially by the fact that the head is the
shape of the head of a fish, distinctly pointed. With the fleas the lower border of the
head comes out in a straight line to join the curve of the upper part. In the Sarco-
psylla lower and upper border of head are both curved.

DlPTERA

The insects of the order Diptera are of great importance medically
in a variety of ways, either by the direct irritation of their bites, by their
transmitting disease directly, as does the common house fly typhoid
fever, or by acting as intermediate or definitive hosts for various
parasites. They are characterized by mouth parts formed for punctur-
ing, sucking, or licking. They present a complete metamorphosis, larva,
pupa, and imago. As a rule, the Diptera have a distinct pair of wings
the second pair being rudimentary.

The order Diptera is usually divided into the following suborders: i. Orthorrha-
pha: Diptera with larvae having a differentiated head. The imago breaks through

352

THE INSECTS

al space

the larval or pupal case by a T-shaped break and has no frontal lunule (an oval sj
just above the root of the antennae). The Orthorrhapha are divided into: a. Nemo-
cera (with long, many jointed antennae) and b. Brachycera (with short antennae).

The Nemocera are generally midge-like insects and have, as a rule, long slender palps
(mosquito). The Brachycera, however, are seldom midge-like. The antennae are
composed of only two or three simple joints with or without style or arista. The palps
are almost always short and never more than two- jointed. 2. Cydorrhapha: larvae
without differentiated head. The imago escapes through an anterior opening and has
a lunule and ptilinum (an inflatable projecting organ just above the root of the an-
tennae). If the halteres are covered by a scale (squama) we have calyptrate Cyclor-
rhapha; if not, acalyptrate. These squamae are large enough in the calpytrate species
to even conceal the halteres when the fly is looked at from above. 3. Pupipara: the
larvae are extruded from the mother and almost immediately begin the pupal life.
Leathery flies with poorly developed wings (Hippobocidae).

The males of flies where the two compound eyes come together above the antennae
are referred to as holoptic, if more or less widely separated as dichoptic. Ocelli are
three single eyes usually, when present, situated in the triangular space between the
compound eyes in the front (the space separating the compound eyes).

The anterior portion of the head which lies below the origin of the
antennae is the face and on each side of the face we have the cheeks
which should be studied as to presence of abundance of hairs. The
antennae which separate the front from the face are of great importance
in classification. In the Muscidae the appearance of a feathery struc-
ture, projecting from the terminal segment of the antennae, and called
the arista, is important. This may be bare or feathered and the
feathering may be only on one side or of one part.

In studying the biting flies it is very important to recognize the anterior, small,
or mid-cross vein. This short transverse rib or vein is the key to wing venation.
Beneath it is the discal cell and it bounds the first posterior cell internally or basally.
The fourth longitudinal vein, which touches the bottom of the mid-cross vein, is of
particular importance as it gives different shapes to the first posterior cell as it runs
along the lower border of this cell. The closed-in discal cell is below the fourth
longitudinal vein. The character of the antennae should also be noted carefully.
The study of the bristles about head, thorax, and abdomen (chaetotaxy) is more
difficult. Anyone taking up the study of flies should carefully note the wings, etc.,
of Musca domestica. By putting a few house flies on moist horse manure in a gauze-
covered bottle the entire metamorphosis may be observed.

Tabanidae

This is the family of horseflies, gadflies, breeze flies or green-headed flies. It
is the most numerous family of the Diptera — there being more than 1000 species.
The females are blood suckers; the males live on flowers and plant juices. The
eyes are usually very brilliant in color, and in the male make up the greater part of
the head.

THE HOUSEFLY

353

They belong to the suborder Orthorrhapha and in the group of short antennae flies
(Brachycera). Five posterior cells are always present.

The antennae consist of three segments. No arista. The epipharynx is tube
like, the hypopharynx has a groove and both are awl-shaped. The pair of maxillae
are serrated and the mandibles lancet like. They have rather coarse maxillary
palps. The labellae are prominent at the extremity of the fleshy labium. They are
thick set flies and rarely show color. The body of the larva has eleven segments
with a small but distinct head. The eggs are deposited in masses on the leaves or
stems of plants about marshy places. The larva is carnivorous.

Tabanus autumnalis. — Is about % inch long; it is dark in color, and has four
longitudinal bands on the thorax. The last joint of the antennae has a crescentic
notch. The wings do not overlap.

Haematopota pluvialis. — In the Hosmatopota there is no crescentic antennal notch,
and the wings overlap. The abdomen is narrower than in Tabanus. The brimp, one
of the Hcematopota, bites man severely.

FIG. 85. — Common housefly (Musca domestica): Puparium at left; adult next,
larva and enlarged parts at right. All enlarged. From circular 71 (by L. O.
Howard), Bureau of Entomology, U. S. Department of Agriculture.

Pangonia beckeri. — The genus Pangonia is characterized by a very long, slender,
and more or less horizontal proboscis.

Chrysops dispar. — Chrysops has three ocelli, in this respect differing from the
genera Tabanus and Hcematopota. The wings are widely separated and spotted.
The antennas of Chrysops are especially long and slender. Chrysops and Hcemato-
pota produce the greatest amount of pain from their bites. The Tabanidoe except
Chrysops are not implicated as intermediate hosts in the transmission of disease. By
their bites, however, they may transmit disease directly, as with anthrax. Two
species of Chrysops, C. dimidiata and C. silacea, have been found to transmit

Filaria loa.

Muscidae

The Muscidce, Sarcophagidae, and (Estridae are calyptrate Cyclorrhapha.
Musca domestica. — The common housefly, Musca domestica, is the best example of
this family.
23

354

THE INSECTS

fourth

The arista is feathered both dorsally and ventrally with straight hairs. The fourtl
longitudinal vein bends down in a rather sharp angle as compared with Stomoxys,
which gives the first posterior cell of the latter rather a fusiform appearance. The
eyes are close together in the male, far apart in the female. The female lays about
125 eggs in a heap preferably in fermenting horse manure. The larva comes out in
about thirty-six hours. Very characteristic are the stigmata decorating the blunt
posterior ends. (See illustration.)

The larval stage lasts seven to ten days and then the barrel-shaped pupal stage

Phlebotomus [Psychodidae]

(Phoridae)

FIG. 86. — Wing venation of Diptera. A, First posterior cell; £, discal cell; b,
mid cross- vein; a, auxiliary vein; C, marginal cell; D, submarginal cell.

is entered upon. This lasts about three days when the adult fly emerges. This is
termed a "coarctate" pupa. The larva shrinks and is surrounded by its old skin
which is termed a puparium. This fly is incapable of biting, the piercing organs
being fused with the labium, but may transmit disease directly, carrying infectious
material from the source, as in faeces, to the food about to be ingested. Their r6le in
typhoid fever is one of immense importance. By reason of its hairy sticky legs,
habits of frequent defecation and constant regurgitation the housefly is an important

TSETSE FLIES

355

agent in the spread of cholera, dysentery, infantile diarrhoeas and tropical ophthal-
mias as well as typhoid.

In the Muscidae the antennae hang down in front of the head in three segments
and have an arista plumose to the tip. The first posterior cell is narrowed. There
are no bristles on abdomen except at tip.

(I) Stomoxys, Hazmatobia and Glossina have a more or less elongated
proboscis adapted for biting. Stomoxys has delicate palpi, shorter than
the proboscis, and arista feathered only on the dorsal side with straight
hairs. Hasmatobia has club-like palpi about as long as proboscis and
arista with hairs dorsally and ventraUy. Glossina has thick set but
not clubbed palpi and an arista feathered on the dorsal side with
branching hairs.

(II) Musca, Callipkora, Chrysomyia, Lucilia, and CordyloMa do
not have a proboscis adapted for biting.

Stomoxys calcitrans. — These greatly resemble the common housefly in size and
shape. They can be easily distinguished by the black, piercing proboscis extend-
ing beyond the head. There are longitudinal stripes on the thorax and spots on the
abdomen. The proboscis on examination will be seen to be bent at an angle near its
base. The palps are short and slender. The wings diverge widely.

The female lays about 60 banana-shaped eggs in horse manure. These hatch
out in three days as larvae which turn into pupae in two or three weeks. After about
ten days the fly emerges. The genus Stomoxys includes vicious biters. This is the
fly which comes into houses before a rain, and which has given the common housefly
the reputation of biting before a rain. Stomoxys may be implicated in transmitting
surra (Trypanosoma evansi).

It assumed great importance as a transmitter of poliomyelitis and
possibly of pellagra a few years ago — views now discredited.

The horsefly (H&matobia irritans) rarely bites man. In these the
palpi are much longer than in Stomoxys, being as long as proboscis.
These palps are also thick and spatulate.

Glossina palpalis. — This is the tsetse fly that is responsible for the
transmission of human trypanosomiasis (sleeping sickness).

The tsetse fly is a small brownish fly about H inch long. The proboscis
extends vertically and has a bulb at its base. The arista is plumose only on the
upper side and the individual hairs are themselves feathered. The wings are carried
flat, closed over one another like the blades of a pair of scissors and project beyond
the abdomen. The most characteristic feature of the tsetse fly is the way the fourth
longitudinal vein bends up abruptly to meet the midcross vein and then curves
downward to run parallel with the third longitudinal vein. In Stomoxys, the wings
separate; in Hcematopota they just meet, and in Glossina they cross. Glossinae bite
chiefly in the daytime.

The tsetse fly is much like Stomoxys, but has a branching of the feathering of the

356

THE INSECTS

arista, long palps, a bulb to the proboscis and a characteristic upbending of the
fourth longitudinal vein to meet the midcross vein. The female deposits her larva
near a shady place upon loose, dry, sandy soil. Moisture and sunlight are not favor-
able for pupal development, the sun being particularly injurious, so that pupae,
buried only an inch deep and away from shade, are killed. This fact has been
utilized in prophylaxis by cutting down the trees. The trouble is that the bush
growth which soon follows is favorable as shade for the pupae.

The female gives birth to a single, yellowish brown, motile larva, which is almost as
large as the mother and which, upon reaching the ground, bores its way into a coarse,
sandy soil for a depth of about 2 inches and then becomes a pupa. The larval stage

FIG. 87. — Insects in which the adult stage is important, (i) Stomoxys calcitrans;
(2) S. calcitrans, larva; (3) Tabanus bovinus; (4) Tabanus larva; (5) Glossina palpalis;
(6) G. palpalis, side view; (7) G. palpalis pupa; (8) Glossina palps and arista.

in the mother lasts about two weeks and the pupal stage in the ground about a
month,

Male and female flies bite and transmit the disease. They bite in the daytime,
usually from 9 A.M. to 4 P.M., and will bite in the sunlight.

With a view to eradication of the disease certain areas have been depopulated, but
upon examining the flies caught in the district a year or more later, infected flies
have been obtained. This would indicate some other reservoir than man. It is
now generally conceded that the trypanosome strain in the antelope is the same as
T. rhodesiense, both being transmitted by G. morsitans. Taute however believes them
different as he not only injected blood containing such trypanosomes into himself,

FLESH FLIES 257

with negative result, but also allowed flies which had fed on antelopes, which were
infective for laboratory animals, to feed on himself, likewise with negative result. It
is a well-known fact that men in good condition are refractory to trypanosome infec-
tion so that this courageous experiment does not prove the antelope strain to be
different from the human one.

One measure that has been proposed is to kill off the big game from a certain area
with a view to depriving the flies of their main source of infection.

The probabilities of an animal reservoir for T. gambiense however is not so well
settled. ^Many think that we may have trypanosome carriers and that such persons
in the enjoyment of health may act as reservoirs of the virus. Koch suggested that
crocodiles were important factors in the life of the tsetse flies and recommended
the destruction of the crocodile eggs.

Glossina morsitans transmits the cattle trypanosome disease, nagana and the
human infection due to Trypanosoma rhodesiense.

Auchmeromyia luteola.— This is an African fly, the larva of which is known as
the "Congo floor maggot," and is a blood sucker. The larva is of a dirty-white
color and about %j inch long. It crawls out at night and feeds on the sleeping native.
This is the only known instance of a blood-sucking larva.

Calliphora vomitoria and Lucilia caesar.— These are flies with brilliant metallic-
colored abdomens, commonly called blow flies in the case of Calliphora and blue-
bottle flies for Lucilia. They deposit their eggs on tainted meat and in wounds.
Many cases of obscure abdominal trouble are probably due to the larvae of these
flies. Intestinal myiasis is undoubtedly of greater importance than has been
thought. The larvae, with hook-like projections anteriorly and a ringed body, can
easily be recognized in the faeces. They have been mistaken for flukes. They
also have a tendency to be attracted by those with ozena and the larvae may develop
in the nostrils. Cheeks bare in Lucilia, hairy in Calliphora.

Chrysomyia macellaria.— This is known as the screw- worm when in the larval
stage. The adult fly resembles the blue-bottle flies. It is distinguished from them,
however, by the presence of black stripes on thorax. These flies are very common
over nearly all North and South America. The eggs which number 250 or more,
when deposited in the nostrils or in wounds, develop into the screw-worm larva,
which may, by going up into the frontal sinus, cause death. These larvae have
twelve segments with rings of minute spines.

Ochromyia anthropophaga (Cordylobia anthropophaga or Tumbu Fly). — This
is an African fly whose larvae develop under the skin of man and animals. It is
known as the Ver de Cayor. The larva resembles the Ver Macaque, is rather
barrel-shaped and beset with small spines. It bores its way into the skin and makes
a lesion like a boil which has a central opening through which the larva breathes.

Sarcophagidae

These are known as "flesh flies." The most important characteristic is the fact
that the arista is plumose up to the mid-point, beyond which it is bare. They are
usually thick set and moderately large flies.

Sarcophaga carnaria. — This is a grayish fly with three stripes on thorax and
black spots on each segment of the abdomen. It is viviparous. The larvae gain
access to nasal and other cavities and there develop. Cases of death have been

358

THE INSECTS

of war

reported. Naturally, the fly deposits its larvae on decaying flesh. In times of
all of these flies become important by reason of "maggots" in the wound. These
larvse are the most common ones in intestinal myiases. The mouth booklets are
strongly curved and separate. Each abdominal segment has a girdle of spines.
The anterior end is somewhat pointed. The hind stigmal plate is in a deep cavity.

CEstridse

The flies of this family are usually called botflies. The mouth parts are almost
vestigial. They have a large head with a somewhat bloated-looking lower portion.
They are often rather hairy. The larvae which develop from the eggs are parasitic
either in the alimentary canal or the subcutaneous tissues.

FIG. 88. — Insects in which the larval stage is important, (i) Chrysomyia macel-
laria; (2) C. larva; (3) Dermatobia cyaniventris larva, early stage (ver macaque);
(4) D. cyaniventris larva, later stage (torcel or berne); (5) D. cyaniventris; (6)
Auchmeromyia luteola; (7) A.luteola, larva; (8) Sarcophaga magnifica; (9) S. magnifica
larva; (10) Anthomyia pluvialis; (n) A. phtvialis larva.

Dermatobia cyaniventris. — These are large, thick-set flies about % inch long,
with prominent head and eyes, small antennae, and a marked narrowing at the
junction of thorax and abdomen. The thorax is grayish and the abdomen a metallic
blue. The larvae are deposited under the skin in various parts of the body. When
the larvae move they cause considerable pain. At first the larva is club-shaped,
but later on it becomes oval. The former is called Ver Macaque, the latter Torcel.

Hypoderma diana.— The larval form of this fly has been reported three times

THE SCREW-WORM 359

for man. It forms tumors under the skin which it is thought may reach this location
by proceeding in some way from the alimentary canal.

In Hypoderma the arista is bare while in Dermatobia the upper border is plumose.

MYIASES

Ver Macaque. — The best known of these myiases is that due to the
larva of a gadfly, Dermatobia cyaniventris.

The larva is at first club-shaped and in this stage is called ver macaque. Later on
it becomes worm-shaped and is then called torcel in Venezuela or berne in Brazil.
The natives of most of the countries where the infection is found have called the
larvae "mosquito worms" or "gusano de zancudo" and they have even incriminated
large mosquitoes belonging to the genus Psorophora as being responsible for the
infections.

Surcouf has noted that these fly larvae have been found cemented to mosquitoes
of the genus Janthinosoma by a glue-like substance. These mosquitoes are vicious
biters and evidently the young larvae escape from the eggs attached to the mosquito
and enter the wound made by the biting parts of the mosquito. Some have thought
that D. cyaniventris deposits its eggs in a glue-like material on the leaves of plants
and that they stick to mosquitoes flying about such plants. From the facts that
these eggs apparently only become attached to this particular mosquito and further
in that the eggs are attached in a constant manner with the hatching end outward it
would seem that the mother fly must in some way seize the mosquito and deposit her
eggs on it. As the larva grows in the subcutaneous tissues of man or other animals
a tumor-like swelling develops with a central orifice, toward which the posterior
extremity of the larva points and through which it takes air into its spiracles.

The swelling somewhat resembles a blind boil and may be as large as a pigeon's
egg-

These gadfly boils tend to break down and discharge a sero-purulent fluid and it is
supposed that the larva, when mature, escapes as a result of the disintegration of the
tumor.

In Brazil they make tobacco juice applications which cause the larva to protrude
and then squeeze it out. The injection of a little chloroform into the larva with a
hypodermic syringe, prior to its extraction with a forceps, makes the process less
painful.

The Screw-worm. — This is the larva of a blue-bottle fly, Chrysomyia
macellaria, which differs from the common blue-bottle fly, Lucilia,
by having three black lines on scutum,

This muscid fly lays 200 to 300 eggs in wounds or orifices having offensive dis-
charges, as from nose, ears, etc. The larvae burrow into the adjacent tissues and
cause frightful destruction of all soft parts. The mature larvae are a little more
than % inch long and have circlets of spines around each of the 1 2 segments.

This infection is especially common in tropical and subtropical America and is
important in animals as well as man.

THE INSECTS

Intestinal Myiases

In the tropics vague intestinal disturbances or violent abdominal
cramping may be brought about by dipterous larvae in the intestinal
canal. The symptoms may be those of a dysentery and may be at-
tended with fever and malaise.

The larvae usually obtain access to the alimentary tract in food taken in by the
mouth. Flies of the genus Sarcophaga are prone to deposit their larvae on food,
especially meat that is somewhat tainted. Other flies, as Musca or Anthomyia,

FIG. 89. — Markings of breathing slits on posterior stigmata of various dipterous
larvae, i. Musca domestica, showing both stigmata; 2. Calliphora vomitoria; 3.
Stomoxys calcitrans. 4. Auchmeromyia luteola; 5. Cordylobia anthropophaga; 6.
Sarcophaga magnifica.

may lay their eggs on food. Flies of the genus Anthomyia tend to lay their eggs on
plants.

It is possible for a fly to deposit its eggs or larvae about the anus
while the man is at stool.

Great care must always be observed to assure one's self that fly larvae, which may
be present in the stool, have not originated from larvae deposited on the stool subse-
quent to its passage.

Aural Myiases

While the larva of Chrysomyia macellaria, known as the "screw-
worm," is the one most frequently reported from the external auditory

MYIASES 361

canal yet many such cases have been connected with the larvae of
Sarcophaga carnaria, Calliphora wmitoria and Anthomyia pluvialis.
These larvae are usually deposited in the auditory canals of those with
otorrhoea.

The symptoms are intense earache, giddiness and possibly convulsions. The
larvae tend to perforate the tympanic membrane. Instillations of 10% chloroform
in milk or the use of oils kill the larvse.

DETERMINATION OF DIPTEROUS LARV.E

There are certain points in the anatomy of dipterous larvae which
must be considered in recognition of the genus or family of the flies con-
cerned in the various myiases. The broad extremity is the posterior
one and the tapering one the anterior. The dark hooklike processes,
which may be in pairs or fused, project from the anterior or head end
and above them are a pair of projecting papillae. The second segment
from the head has on either side projecting hand or fan-like structures
with varying numbers of terminal divisions, 4 to 40 or more. These
are the anterior spiracles.

The large terminal segment has on its posterior surface two chitinized plates with
3 slits of various architecture in each. These are the posterior stigmal plates and
are the structures we pay particular attention to in identification. In the early
larval stages there is only one slit; in the second stage there are two. It is only in
the fully developed larval stage that we note the characteristic 3 slit stigmal plates.
The presence or absence of a rounded protuberance or button at the base of each
stigmal plate should be looked for. The area carrying the stigmal plates may be
sunken to form a pit.

KEY TO LARV2E OF THE MYIASES (BANKS)

1. Body with lateral and dorsal spinose processes Homalomyia.

Body without such processes 2

2. Body ending in two fleshy processes; rather small species 3

Body truncate or broadly rounded at end 4

3. Processes bearing the stigmal plates; body about 5 mm. long. . . . Drosophila.
Processes not bearing the stigmal plates; body 10 mm. or longer . Piophila.

4. But one great hook; posterior stigmal plates with winding

slits; no distinct lateral fusiform areas; tip of body with

few if any conical processes Muscinae.

With two great hooks; slits in the stigmal plate not sinuous 5

5. No tubercles about anal area; no distinct processes around

stigmal field 6

Distinct tubercles above anal area; often processes around

stigmal field; lateral fusiform areas usually distinct 7

362 THE INSECTS

6. Stigmal plates on black tubercles; lateral fusiform areas distinct. Ortalidae
Stigmal plates barely if at all elevated; lateral fusiform areas

indistinct; stigmal plates often contiguous or nearly so;

slits long and subparallel Trypetidae.

7. Slits in stigmal plates rather short, and arranged radiately. ... 8
Slits slender and subparallel to each other 9

8. Two tubercles above anal area; stigmal field with distinct

processes around it Anthomyiidae.

Four or more tubercles above anal area; slits of stigmal plates

usually pointed at one end Muscina.

9. A button to each stigmal plate; slits rather transverse to body. . . Calliphorinae.
No button to stigmal plates, slits of one plate subparallel to

those in opposite plate; plates at bottom of a pit Sarcophagidae

CHAPTER XXI
THE MOSQUITOES

MOSQUITOES (Culicidae) are of the greatest importance medically,
not only from their influence upon health in general by reason of inter-
ference with sleep and possibly from direct transmission of disease, but,
more specifically, they are the only means by which it at present appears
possible to bring about infection with such diseases as yellow fever,
malaria, filariasis, and possibly dengue. In addition, many diseases
of animals are transmitted by mosquitoes.

The Culicidse differ from all other Diptera in having scales on their
wings and generally on head, thorax, or abdomen.

To identify a mosquito, examine a wing and note the scales; also note the presence
of two distinct fork cells and, in addition, that the costal vein passes completely
around the border of the wing, making a sort of fringe with its scales. Mosquitoes
undergo a complete metamorphosis, there developing from the egg a voracious,
rapidly growing larva; next, a nongrowing, nonf ceding stage— the pupa or nymph.
In this latter the head and thorax are combined is an oval body, from the back
of which projects the siphon tubes; and tucked in ventrally is a small tail-like
appendage.

The fully developed insect emerges from the pupa.

The Culicidae belong to the suborder Nematocera. These have long articulated
antennas and include four families: Culicidae, Chironomidae, Simulidae and Psycho-
didaj.

The principal mosquito-like, blood-sucking Diptera which are
frequently mistaken for mosquitoes— none of which have scales on their
wings — are the following:

1. Chironomidae or Midges. — The blood-sucking species of Chironomidae, which
are found in- most parts of the world, belong chiefly to the genus Ceratopogon. These
midges are of very small size, about ^2 mch long, are able to get through netting
and, usually being in swarms, they are exceedingly troublesome. The antennae
have thirteen joints and the wings are shorter than the abdomen and have only
longitudinal veins. One of the midges, the "jejen" of Cuba, is a great scourge,
its small size enabling it to enter eyes and nostrils. The larva of Chironomus is a red
worm-like creature; the pupa has a tufted head.

2. Simulidae or Buffalo Gnats. — These are small blood-thirsty insects only about
% inch in length. The thorax is humped, the legs are short and the proboscis

363

364

THE MOSQUITOES

short and inconspicuous. The antennae have n joints but are rather shoi
One species, the S. damnosum, known, by the natives of Uganda as "Mbwa," is
greatly dreaded; its bites causing swellings and sores. Sambon has considered
Simulium reptans as the transmitting agent of pellagra.

3. Psychodidse or Moth Midges. — These are small, hairy, slender midges, with
long legs and a short proboscis. The antennae are long, hairy and consist of 12 to 1 6
joints. Palpi four jointed. They are only about J^2 inch in length. The hairy
wings have numerous longitudinal veins. Some, as Phlebotomus, have an enlongated
proboscis and are vicious blood suckers.

FIG. 90. — Mosquito-like insects belonging to families Chironomidae, Simulidae
and Psychodidae. (i) Phlebotomus papatasii; (2) P. papatsii (natural size); (3)
P. papatasii (larva) ; (4) P. papatasii larva (natural size) ; (5) Ceratopogon pulicaris;
(6) C. pulicaris (natural size); (7) Chironomus larva; (8) Attitude of a Simulium;
(9) Simulium reptans; (10) Larvae of Simulium.

At present, of the genera of the three families of midges, only Phle-
botomus is known to transmit disease. P. papatasii transmits phle-
botomus fever in the Balkans. P. minutus is the host at Aden.
Another species, P. perniciosus can transmit the disease. These moth
midges are 2 mm. in length and have the body densely covered with
long yellow hairs. The second longitudinal vein has three distinct
branches. The antennae have 16 constricted joints and the proboscis
is as long as the head. The species of Phlebotomus are separated by

PHLEBOTOMUS 365

slight variations in wing venation, palpal lengths, etc., thus the second
segment of palpi of P. papatasii is a little longer than the third one
while with P. perniciosus these segments are of equal lengths. In
P. minutus the second segment is only half the length of the third.
The insect lays about 40 eggs in damp dark places. The period of
metamorphosis from egg to insect is about one or two months,
according to temperature.

Phlebotomus larvae die out in dry soil and very wet earth is un-
favorable. Moderate moisture and protection from light seem neces-
sary for their development. The remains of dead insects also seem
to make good breeding places. It is in cracks of old damp brick
or stone walls that the female most often deposits her eggs. Caves
are also selected. Blood seems necessary for the fertilization of the
eggs but lizard blood seems more common in the stomach of P. minutus
than human blood. They have also been observed to feed on other
reptilian bloods. The female insect has been kept alive in captivity
up to forty-six days.

Culicidae. — Mosquitoes have three main parts of the body — the head,
the thorax, and the abdomen. On the head, the space behind the two
compound eyes is called the frons, in front, and the occiput posteriorly.

The nape is back of the occiput. The bulbous prolongation of the frons which
projects over the attachment of the proboscis is the clypeus. The clypeus is hairy
in the Culex; scaly in Stegomyia. The proboscis is straight in all mosquitoes of
importance medically. It consists of a fleshy, scaled, gutter-shaped portion be-
neath, known as the labium, which terminates in two hinge-joint processes — the
labella. At the end of the labium is a thin membrane (Button's membrane). It
is through this that filarial embryos are supposed to pass on their way from the
interior of the labium to enter the person bitten. The labium may be considered
as the sheath of a knife, holding and protecting the slender, blade-like penetrating
organs. Lying in this groove we have, from above downward, the horseshoe-
shaped labium -epipharynx, the undersurface of which is open. This when closed
by the underlying hypopharynx forms a tube through which the blood is sucked
up by the mosquito. In the hypopharynx, which somewhat resembles a hypoder-
mic needle, is a channel, the veneno-salivary duct. It is down this channel that
the malarial sporozoite passes. There are two pairs of mandibles and two pairs
of maxillae on either side of the hypopharynx — the mandibles above and the maxillae
below. The serrations of the maxillae are coarser than those of the mandibles.
The sensory organs, the palps, lie on either side of and slightly above the proboscis.
These are of the utmost importance in differentiating mosquitoes and must not
be confused with the antennae, which are attached above the palpi and at the sides
of the clypeus. These antennae are of importance in distinguishing the sex of the
mosquito.

The thorax is largely made up of the mesothorax, at the posterior margin of

366

THE MOSQUITOES

•r trilo-

which is a small, sharply defined piece, the scutellum; this may be smooth or
bed. Underneath and posterior to the scutellum is the metanotum; the metano-
tum is bare in Anophelinae and Culicinae, has hairs in Dendromyinae and scales in
Joblotinae.

There is a pair of wings attached to the posterior part of the mesothorax and,
more posteriorly still, a pair of rudimentary wings (halteres) attached to the
metanotum.

The wing venation is important. The costa shows as a stout rib
or vein bordering the upper side of the wing and running around the.
apex and lower border.

FIG. 91. — Anatomy of the mosquito. No. 7 shows various types of scales.

Below, it has a fringe which may show spots. The location of the spots in the upper
part of the costa of anophelines is of great value in differentiating species. Beneath
the upper costal border the subcostal vein runs to join the costa at some distance
within the apex. The apex is the free end of the wing and the base that attached
to the thorax. Running parallel to the subcosta, but reaching the apex, is the ist
longitudinal vein. Below that is the 2d longitudinal vein which forks to make
the ist fork cell or ist submarginal cell. The 3d longitudinal takes origin at the junc-
tion of the supernumerary and midcross veins. The 4th longitudinal divides to
form the 2d fork cell (zd posterior cell). The 5th and 6th longitudinal veins
arise from the base of the wing and run to the periphery.

ANATOMY OF THE MOSQUITO

367

The three pairs of legs are attached to the thorax.

Each leg has 9 parts. The two short ones are the basally placed coxa and the
small trochanter attached to it. Then come the long femora, tibia and metatarsi
with the four segments of the tarsi terminally. The last tarsal segment ends in two
claws, which in the female may be simple or uni-serrated.

There are nine segments in the abdomen. The genitalia arise from the terminal
segments as bilobed processes. In the male there is a pair of hook-like appendages
or claspers, between which, and ventrally situated, are the harpes, also a pair of
chitinous processes.

FIG. 92. — Distinguishing characteristics of mosquito larvae and fly antennas
Siphon tubes of i, Stegomyia, 2, Culex, 3, Tcsniorhynchus; mental plates of 4, Tcenio-
rhynchus, 5, Stegomyia, 6, Culux; larval antennae of 7, Culex, 8, Stegomyia, 9, Anoph-
eles; antennae of 10, Muscidae, n, Tabanidae, 12, Simulidae, 13, Sarcophagidse.

In considering the question of the possible danger which might arise from the
introduction of a case of yellow fever, malaria, or filariasis, it would give the greatest
information if mosquito ova were at hand so that we could by watching the develop-
ment from egg to larva, pupa, and insect, have all the points from which to decide as
to the genera developing in the given locality. It is generally a very easy matter
to dip out large numbers of larvae from the pools and having noted the characteristics
of the larvae, to do the same when the pupae develop; so that we have only to verify
our identification when the insect emerges from the pupa.

THE OVA

The egg raft of Culex, containing about 250 ova, is quite perceptible on the surface
of the water as a black, scooped-out mass, about ^ inch in length. The eggs

368

THE MOSQUITOES

are set vertically in the raft. The eggs of the Stegomyia are laid singly and have
a pearl-necklace-like fringe around them.

The Anophelinae eggs are oval in shape with air-cell projections from either side.
They are laid in triangle and ribbon patterns. The markings of these air cells vary
and have been used for differentiation. The length of time of the egg stage varies
according to temperature and other conditions — one to three days for Stegomyia
and two to four days for Anophelina. The Anophelinae are more difficult to raise
than Culex or Stegomyia.

LARV.E

There are two great classes of larvae — the siphonate and the asiphonate.
latter are always Anophelinae.

The

STIGMATlC

" UATERAL. ABDOM. HXVIRS

ANTENNA

MOUTH BRU

FIG. 93. — Asiphonate larva. Anopheles. 2. Siphonate larva. Stegomyia.

The Culicinae larvae have a projecting breathing tube at the posterior extremity
which is called a respiratory siphon. This projects off at an angle from the axis of
the body, the true end of which terminates in four flap-like paddles. If you divide
the length of the siphon by the breath, you get what is known as the siphon index.
In Culex the siphon is long and slender, in Stegomyia it is short and barrel-shaped.
When at the surface the Culex larva has its siphon almost vertical and the body at an
angle of about 45°.

MOSQUITO LARV2E

The Stegomyia larva hangs more vertically. As a rule, the hairs proceeding
from the sides of Culex larvae are straight and the head relatively large. There are
also no palmate hairs along the sides.

The Anophelinae larvae have a small head which is capable of being twisted around
with lightning-like rapidity. They are darker in color and have no siphon; float
parallel to the surface of the water; have long lateral branching hairs, and on the
sides of each of the five or six middle abdominal segments they have a pair of
palmate hairs. These palmate hairs are supposed to aid them in keeping their posi-
tion on the surface of the water. The larva are usually
called "wrigglers." The duration of the larval stage is
from one to two weeks, according to the temperature.

It has been proposed to use larval character-
istics in differentiating species but as the larva
moults about three times and as the hairs or
spines of the exo-skeleton of these different larval
stages vary in number and appearance such a
scheme has not met with general approval.

THE PUP.E

The pupa of the mosquito is an obtected one
there being only a closely applied chitinous coating
covering it; it does not have a puparium as does
the coarctate pupa of the house fly. The mos-
quito pupa is lighter than water while the larva
is heavier.

Pupae have a bloated-looking cephalo-thorax and a
shrimp-like tail — the latter the abdomen. Very impor-
tant in examining them with a lens is to note the char-
acteristics of the siphon tubes which project from the
dorsal surface. These siphons are long and slender in
Culex and project from the posterior portion of the head
end. In Anophelinae they are broadly funnel-shaped and arise from the middle
of the head end. The siphon of the Stegomyia is triangular.

The bulbous end of the Culex nymph is more vertical than the horizontally placed
cephalo-thorax of Anopheles. The duration of pupal life is short — only one to three
days. At the end of this time the pupa comes to the surface and straightens out.
The integument then splits dorsally and the perfect insect emerges. It dries its
wings for a time on its raft-like pupal skin and then flies away.

From the above it will be seen that the stages in the metamorphosis of the mos-
quito take about two weeks: one to three days for egg stage; seven to ten days for
larval stage, and two to three days for pupal stage.

FIG. 94. — Pupae: i.
Culex; 2. Anopheles;
3. Aedes calopus.
(After Howard.} From
P. H. Reports.

24

370

THE MOSQUITOES

DISSECTION OF THE MOSQUITO

The easiest way to secure a mosquito for dissection is to use an ordinary plugged
test-tube. Slipping the open end of the test-tube over the resting mosquito, by a
slight movement, the insect will fly toward the bottom. Then quickly insert the
plug. If it is not desired to study the scales, the best way to kill the mosquito is by
striking the tube sharply against the thigh. If it is also desired to study the scale
characteristics it is better to put a drop or so of chloroform on the lower part of the
cotton plug. The vapor falls to the bottom of the tube and kills the mosquito. Take
the mosquito out, pull off legs and wings, and then place the body in a drop of salt

FIG. 95. — Heads of mosquitoes: i and 2, male and female Cttiex pungens', 3 and 4,
male and female Anopheles', 5 and 6, male and female A'edes calopus. (After Stitt.)
From P. H. Reports.

solution on a slide. It has been recommended to smear the surface of the slide with
bile, wiping off the excess, before commencing the dissection in the salt solution.
Then hold the anterior end of the thorax by pressure of a needle. With a second
needle in the other hand, gently crush the chitinous connection between the sixth and
seventh segments of the abdomen. Then holding the thorax firm, steadily and
gently pull the last segments in the opposite direction. If this is done properly, a
delicate gelatinous white mass will slowly float out in the salt solution. One should

CULICINE AND ANOPHELINE MOSQUITOES 371

be able to secure the alimentary canal as far up as the proventriculus, which is just
anterior to the stomach, the part in which the malarial zygotes develop. Proceeding
from before backward, we have the proventriculus, which is a sort of muscular ring
at the opening of the stomach or mid-gut and marks the separation of the stomach
from the cesophagus. Opening into the lower part of the oesophagus are the
cesophageal diverticula or crops, which are food reservoirs. Occasionally in a
dissection we pull out these structures which are three in number.

Leading from the stomach we have the hind-gut, which ends in the rectum.

This has a posterior dilatation or rectal pouch which usually has three or four
rather marked anal papillae.

Taking origin at the posterior end of the stomach and festooning the hind-gut
are five longitudinal tubes — the Malpighian tubules. These are characterized by
large granular-like cells with a prominent refractile nucleus. They are regarded as
the renal structures. It is in these tubules that the embryo of the Filaria immitis
of the dog develops. In the female mosquito, the parts withdrawn may seem to be
largely made up of the white oval ovaries. These are connected with the sperma-
thecae, in which the spermatozoa are stored after fecundation by the male. In the
male the testicles are quite distinct. Next to the examination of the stomach for
zygotes, which appear as wart-like excrescences on its outer surface, the most
important structures are the salivary glands, where the malarial sporozoites are found.
The easiest way to dissect out the salivary glands is to press down firmly, but gently,
on the anterior part of the thorax, and then with the shaft of a second needle,
pressing on the head to gently draw the head away from the thorax, so that by this
expression and traction movement you extract them with the head segment. They
are very minute and are to be told by their exceedingly highly refractile appearance.
To examine for sporozoites cover the glands protruding from the neck with a cover-
glass and search with a one-sixth objective for narrow, curved bodies in the sub-
stance of the glands. If present try to smear out the glands between the cover-glass
and slide by pushing the cover-glass along, then withdrawing the cover-glass, dry
quickly and stain the smear on slide or cover-glass with Wright's stain.

The sporozoites are narrow falciform bodies about 12/1 in length, with a central
chromatin dot.

A matter about which there is dispute is as to whether the salivary glands com-
municate with the alimentary canal. Theobald states that there is no connection
between them.

DIFFERENTIATION OF CULICINE AND ANOPHELIN^E

It is impossible even for an entomologist to differentiate mosquitoes
without recourse to elaborate keys and tables. It is a comparatively
easy matter, however, to decide as to whether the mosquito is a prob-
able malaria transmitter or not.

While certain characteristics of the male are used to separate the .Edina; from other
subfamilies, yet it is only with the female that we concern ourselves in differentiating
the Culicinaj from the Anophelinae. Therefore, it is first necessary to distinguish

372

THE MOSQUITOES

the male from the female. If the antennae have not been torn off, this can be decided
by the highly adorned plumose antennae of the male, those of the female being
sparsely decorated with short hairs. The palpi of the Anophelinae tend to be
clubbed, while those of the Culex are straight. If the antennae have been broken off,
look for the claspers at the end of the abdomen.

Male mosquitoes do not feed on blood but on fruits and flowers in-
stead. The puncturing parts of the male are not sufficiently resistant
to penetrate the skin.

Having determined that the insect is a female, we then proceed to place it either
in the subfamily Culicinae or Anophelinae by a study of the relative length of the
palpi to the proboscis. If the palpi are shorter than the proboscis, it belongs to the
Culicinae; if as long or longer, to the Anophelinae. The palpi of the female
Megarhininae are also long, but the proboscis is curved.

Having settled on the subfamily, we separate the genera by con-
sidering such points as character and distribution of scales on back of
head, wings, thorax, and abdomen ; banding of proboscis, legs, abdomen,
and thorax, shape of scales on wings, and location of cross veins.

FIG. 96. — Resting posture of mosquitoes: i and 2, Anopheles; 3 Culex pipienes.
(After Sambon.) From P. H. Reports.

Anophelinae show abundant upright forked scales on occiput. The mesothorax
shows sparse hairs or scales with a smooth scutellum. As a rule, the wings are spotted
(dappled) and the location of these spots give the best clue to the different species of
the genera. With the exception of Bironella the first submarginal cell is large. This
cell is longer than the second posterior one.

In the resting position Culex allows the abdomen to droop, so that it is parallel
to the wall. The angle formed by the abdomen with head and proboscis gives a
hunchback appearance.

Anopheles when resting on a wall goes out in a straight line at an angle of about
45°. It resembles a bradawl.

Classification

There are four subfamilies of Culicidae, differentiated according to
the palpi:

MALARIAL TRANSMITTERS

373

1. Palpi as long as proboscis in females; proboscis
straight. Anophelina.

2. Palpi as long or shorter than proboscis in females;
proboscis curved. Megarrhinina.

3. Palpi shorter than proboscis in females. Culicina.
Palpi shorter than proboscis in male and female.

i . Scales on head only; hairs
on thorax and abdomen.

Palpi as long or longer
than proboscis in male.

The important ones from a medical standpoint are the Anophelinae
and Culicinae.

Anophelinae

Scales on wings, large and lanceolate. A nopheles.
Palpi only slightly scaled.

Wing scales small and narrow and lanceolate.
Myzomyia. Only a few scales on palpi.
Large inflated wing scales. CydoUppteron.
2. Scales on head and thorax f
(narrow curve scales).
Abdomen with hairs.

.?

1. Abdominal scales only on ventral surface.
Thoracic scales like hairs. Myzorhynchus.
Palpi rather heavily scaled.

2. Abdominal scales narrow, curved or spindle-
shaped. Abdominal scales as tufts and dorsal

patches. Nyssorhynchus.

3. Abdomen almost completely covered with scales
and also having lateral tufts. Cellia.

4. Abdomen completely scaled. Aldrichia.

i. Wing scales small and lanceolate. Pyretophorus.

Scales on head and thorax
and abdomen. Palpi
covered with thick scales.

NOTE. — Of the above genera only CydoUppteron and Aldrichia are unproven
malarial transmitters.

The following species of anophelines selected from the different
genera are important transmitters of malaria.

Anopheles maculipennis. — Wings with four spots located at bases of both forked
cells and of second and third longitudinal veins. No costal spots. Palpi yellowish
brown and unbanded. Legs unbanded.

Myzomyiafunesta. — Wings with four yellow spots on a black costa and two black
line spots on third longitudinal vein. Palps with three white rings. Proboscis un-
banded. Legs with faint apical bands.

Pyretophorus costalis.— Costa black with five or six small yellow spots. Palps
with two narrow white bands and white tip. Femora and tibias with yellow spots.
Apical tarsal bands.

Myzorhynchus pseudopictus. — Black costa with two pale yellow spots. Wing
fringe unspotted. Black palps with four pale bands. Apex of palps white.

Nyssorhynchus fuliginosus. — Black costa with three large yellow spots.
Numerous black dots on the longitudinal veins. Palpi black with white tip and two
narrow white bands. Last three hind tarsal segments white.

Cellia argyrotarsis. — Black costa with two distinct and several smaller white spots.

374

THE MOSQUITOES

i . Posterior cross vein nearer
the base of the wing than
the mid-cross vein.

Dark brown palps with two narrow bands and a white tip. Legs with last three
hind tarsal segments white.

The Megarhinin® are of no importance medically.

The genus Megarhinus has the following characteristics:

1. Large mosquitoes with brilliant metallic coloring. (Elephant mosquitoes.)

2. Long, curved proboscis.

3. Caudal tufts of hairs on each side of abdomen.

The JEdinae are not known to play any r61e in transmission of diseases. This
subfamily is characterized by having the maxillary palpi much shorter in both males
and females than the proboscis.

One genus Sabethes is very characteristic, owing to dense paddle-like scale struc-
tures on two or more legs.

Differentiation of Culicinae Genera

1. Proboscis curved in female. Psorophora.

2. Proboscis straight in female.

A. Palps with three segments in the female.

(a) Third segment somewhat longer than the
first two. Culex.

(b) The three segments equal in length. Stego-
myia.

B. Palps with four segments in the female.

(a) Palps shorter than the third of the proboscis.
Spotted wings. Theobaldia.

(b) Palps longer than the third of the proboscis.
Irregular scales on wings. Mansonia.

C. Palps with five segments in the female. Tcenior-

hynchus.

2. Posterior cross vein in line with mid-cross vein. Joblotina.

3. Posterior cross vein further from base of wing than mid-cross vein. Muddus.

Of the Culicinae the genus Stegomyia is of importance on account
of yellow fever. The totally efficient hosts for filariasis (filarial embryos
found in the thorax and proboscis) are chiefly among the genus Culex.
The genera Mansonia and Taniorhynchus may also transmit filariasis.
Some think the Anophelinae genera Cellia and Myzomyia may transmit
filariasis as well as malaria.

The genus Culex is implicated in dengue.

Stegomyia. — This is the most important culicine genus. These are mosquitoes
with silver markings. The head, entirely covered with flat scales, has also some
upright forked scales. Scutellum has dense flat scales. S. calopus is deep blackish-
brown with two thoracic parallel lines with curved silver-white lines outside (lyre
marking). Banding of thorax, abdomen, and legs.

S. calopus bites only at night after the first feeding. The first meal of blood
however may be taken in the daytime. To become infected it must take blood
from a yellow-fever patient in the first two or three days of the disease. After
sucking the blood of a yellow-fever patient the mosquitoes cannot transmit the

MALE AND FEMALE MOSQUITOES

375

• ^

FIG. 97.— Anopheles maculipennis FIG. 98.— Aedes calopus, male. From
(quadrimaculatus} , male. (After Cas- P. H. Reports.

tellani and Chalmers.} From P. H.
Reports.

FIG. 99. — Anopheles maculipennis FIG. 100. — Aedes calopus, female. From
(quadrimaculatus}, female. (Castellani P. H. Reports.

and Chalmers, after Austen.} From
P. H. Reports.

376

THE MOSQUITOES

disease by biting a nonimmune to yellow fever for a period of twelve days. After
this time the mosquito remains infective for its life — in one instance fifty-seven days.

S. sGutdlaris has a single silver stripe down the center of thorax. Mosquitoes
of this genus are often called "Tiger mosquitoes." The larvae have short, barrel-
shaped siphons. They breed particularly in receptacles about the house.

S. pseudoscutellaris, which resembles S. scutdlaris, but has white bands only, at
the sides of the abdominal segments, is thought to transmit filariasis in Fiji.

Culex. — Male palpi long and acuminate. Head has narrow curved and upright
forked scales. Laterally, flat scales. C. fatigans supposed to carry dengue as well
as Filaria bancrofti. It also transmits Proteosoma of birds, the life history of which
in this mosquito paved the way to the epochal discoveries in connection with malarial

FIG. 101. — Culex pungens, male. (After
Howard.) From P. H. Reports.

FIG. 102. — Culex pungens, female. (After
Howard.} From P. H. Reports.

transmission by anophelines. This is a brown mosquito with pale yellow banding
of each abdominal segment. The legs are brown except for the coxae and femora.

Theobaldia. — These Culicinae have spotted wings resembling Anophelinae.
These spots are due to aggregations of scales, not to dark scales. Male palps are
clubbed (like Anopheles).

Mucidus. — This genus has a mouldy look from long twisted gray scales. The
legs are densely scaled.

Mansonia. — This genus is characterized by broad flat asymmetrical wing scales.
As the wing scales are brown and yellow the wings are mottled.

Grabhomia. — Wings have pepper-and-salt appearance with short fork cells.

Taniorhynchus. — This genus is characterized by dense wing scales, which are
broadly elongated with truncated apex.

Acartomyia, — Much like Grabhamia, but scales of head give ragged appearance.
Male palpi clubbed.

A. zammittii was supposed to be concerned in Malta fever, but it is known now
that transmission is by medium of milk of infected goats.

CHAPTER XXII
POISONOUS SNAKES

SNAKES belong to the class Reptilia and the order Ophidia. The
two families to which poisonous snakes belong are the colubrine snakes
(Colubridae) and viperine snakes (Viperidae).

Of the Colubridse the Hydrophinae or sea-snakes with rudder-like compressed tail
and the Elapinae with round tails are most important.

Many of our harmless snakes such as the garter-snake and blacksnake belong to
the Colubridae.

The cobras belong to the subfamily Elapinae and are best known by a neck-like
expansion or hood. The only poisonous colubrine snakes in the United States are
the beadsnake (Elaps fuhius) often called the Florida coral snake, and the sonoran
coral (Elaps euryxanthus).

The beadsnake is black with about seventeen broad crimson bands, which bands
are bordered with yellow.

Although small, they are very venomous. The upper jaw has anteriorly grooved
fangs, which appendages are not present in the nonpoisonous coral snakes, these
latter having teeth in the upper jaw so that the wound shows four rows of punctures
instead of two rows and one larger puncture on each side to mark the entrance of the
fangs.

In Asia there are many important poisonous colubrine snakesj the cobra (Naja
tripudians), the King cobra (Naja bungarus) and the Kraits (Bungarus fasciatus).

All of the Australian poisonous snakes are colubrines.

The Viperidae which are characterized by a triangular head and tubular poison
fangs are the most important poisonous snakes in America. The rattlesnake
(Crotalus), the copperhead (Ancistrodon contortrix), and the water moccasin
(A . piscivorus) are widely distributed in the United States.

There are many harmless snakes which more or less resemble these "Pit Vipers"
as the rattlers, moccasins, and copperheads are called. This term refers to a deep
hole or pit found on the side of the head between the nostril and the eye. It is a
blind sac.

Some divide the Viperidae into the Crotalinae, which possess the pit and the
Viperinae which do not have this structure.

The poison fangs are grooved or perforated and connected with the poison glands
which resemble salivary glands and may be almost an inch in length in large snakes.
The tongue is slender and forked and is a tactile organ.

The jaws are remarkable for their great extensibility, not only vertically, but
laterally, by the .ligamentous connections of the two halves of the mandible or lower
jaw.

377

378

POISONOUS SNAKES

As the fangs are directed backward it is necessary for the snake when striking
to open widely the jaws and bend back the neck. The fangs are then brought
forward and erected by the spheno-pterygoid muscles. The snake bite is a com-
bination of bite and blow. The functional fangs of colubrine snakes however are not
mobile.

In addition to the possession of the pit, these vipers have a more or less trian-
gular head and in particular a single row of large scales on the undersurface posterior
to the vent (anus), while the harmless snakes show an elongated oval head and two
rows of large ventral scales posterior to the vent.

FIG. 103. — i, Single row of scales posterior to vent (poisonous snakes — water
moccasin); 2, double row scales of harmless snake (Natrix); 3 and 5, side and dorsal
view of head of pit viper; 4 and 6, side and dorsal View of head of harmless snake
(Natrix); 7 and 9, bite puncture and skull of Elaps; 8 and 10, bite puncture and skull
of harmless snake.

In examining the wound made by a snake the two punctures of the
fangs indicate the bite of a poisonous snake. If these fang puncture
points are far apart it shows that a large snake, and probably one
capable of injecting a greater amount of venom has given the bite.

When a snake strikes the fangs move from the horizontal to the erect position,
the mouth being widely open. When the fangs enter the jaws close and pressure
is exerted on the poison glands so that the venom pours out.

The amount of venom varies with the size and condition of the snake, an adult
cobra yielding about i c.c.

SNAKE VENOM

379

The cobra, after having bitten, remains attached for a short time while the
daboia strikes with the greatest rapidity and immediately releases itself.

Cobra and krait bites (colubrine snakes) produce more or less
similar symptoms such as paralysis of articulation with nausea and
vomiting and later paralysis of the respiratory apparatus. There is
only an insignificant reaction at the point of bite.

The venom is mainly neurotoxic, causing death by paralysis of cardiac and re-
spiratory centers. Cobra venom is also very haemolytic. This hasmolysin is acti-
vated by the normal complement of the serum of the animal poisoned, the hsemolysin
as contained in the venom not being toxic when alone. Lecithin also has the
property of activating the haemolytic amboceptor of venom.

In rattlesnake bites (viperine snakes) there is marked pain at the
site of the wound with much swelling and haemorrhagic infiltration.
The swelling and petechial mottling spread up the limb from the point
of entrance of the venom. Cold sweats, nausea, weak heart, and syn-
cope are common

Rattlesnake venom is active chiefly on account of its haemorrhagin or rather
endotheliolysin, which destroys the endothelial lining of blood-vessels.

Venoms may also contain proteolytic ferments Which may account for the softening
of muscles in snake-bite cases. The toxic effect of the venom takes place without an
appreciable incubation period, hence different from true toxins.

The most venomous snakes seem to be the sea-snakes (Enhydrina) . This venom
is almost entirely neurotoxic.

The tiger snake of Australia is almost equally venomous and the krait (B. cceruleus)
next. The rattlesnake is about one-fifth as venomous as the krait.

Certain venoms greatly increase the coagulability of the blood so that intravas-
cular thromboses may occur. It is chiefly with the venoms of Daboia and Bun-
garus that such thromboses are likely to occur and this accounts for the almost
instantaneous death which at times results from bites of such snakes.

The nonspecific treatment of snake-bite poisoning is i. by applying a tight ligature
above the site of the bite. The ligature, which should preferably be a rubber band,
is to be applied about a single bone extremity, not about one with two supporting
bones. 2. The making of deep incisions about the fang punctures and thorough
irrigation with a strong solution of potassium permanganate. Rogers has recom-
mended that the punctures be enlarged with a lancet and the resulting wound packed
with crystals of permanganate.

Recently Bannerman has shown that a dog bitten by a cobra cannot be saved
by free incision and the rubbing in of permanganate crystals. It may however be
saved by the immediate injection of 10 c.c. of a 5% solution of permanganate, but
not if two minutes has elapsed. Bites from the daboia are fatal, however the per-
manganate be applied.

He therefore does not consider the permanganate treatment of any practical
value. Rogers thinks that Bannerman's experiments with dogs do not give a true

380 POISONOUS SNAKES

idea of the value of permanganate because he has had success in experimenting with
cats and because it has saved human lives. Chromic acid injections (i%) have also
been recommended.

Internally alcohol does not seem to be of any value, in fact many of the deaths
have been attributed to excessive ingestion of whiskey. Strychnine in large, almost
poisonous doses, was highly recommended in Australia but the statistics seem to
make the value of this remedy doubtful.

Antivenins. — The active agents of snake venoms may be either of the nature of
haemorrhagins, neurotoxins, or fibrin ferments. In colubrine snakes the neurotoxin
vastly predominates while with the viperines it is the hsemorrhagin. Certain
Australian snakes contain all three bodies in about equal proportion while with
the rattlesnakes of America it is almost entirely the haemorrhagin which causes
the poisoning. The Elaps of Florida is a colubrine snake and its venom is neurotoxic
in nature.

The cause of death in colubrine snake bites is chiefly from paralysis of the respira-
tory centers while with the Pit Vipers it is chiefly from haemorrhages in the vital
organs. Antitoxins have been prepared against both viperine and colubrine venoms
and these are specific, thus a colubrine antivenin will not be of value against a
viperine bite. Antivenins should be administered either intravenously or intra-
muscularly. The amounts recommended for injections to neutralize a fatal dose
of snake poison vary from 100 to 300 c.c. of the antivenin serum. There is no
accurate standardization.

PART IV

CLINICAL BACTERIOLOGY AND ANIMAL PARA-

SITOLOGY OF THE VARIOUS BODY

FLUIDS AND ORGANS

CHAPTER XXIII

DIAGNOSIS OF INFECTIONS OF THE OCULAR REGION

IT is advisable before taking material for cultures or smears to
cleanse the nasal area of the eye-lids, and especially about the caruncles,
with sterile salt solution. Then, by gently pressing on the lids, we may
be able to get pure cultures of the organism causing the infection.
Normally, we may find in the region of the caruncles various skin
organisms, especially staphylococci, giving white colonies.

The xerosis bacillus and white staphylococci may be considered normal findings
in the conjunctival sac. Streptococci and pneumococci have also been reported
from apparently normal conjunctival secretions.

A small particle of sterile cotton, wound on a toothpick, with the aid of a sterile
forceps, makes an excellent swab for obtaining material for smears; the same may
first be drawn over an agar surface in a Petri dish in a series of parallel lines of in-
oculation before making the smears on slide or cover-glass.

When there is a considerable discharge, a capillary pipette, with a rubber bulb,
may be used to draw up sufficient material for cultures and smears. Be sure to
round off the end of the pipette in the flame and not to use a very fine capillary
tube.

In conjunctival cultures, plates of glycerine agar, blood agar, or agar
plates smeared with blood are to be preferred, as the Gonococcus and
Koch- Weeks bacillus will only grow on blood or hydrocele agar. The
diphtheria and xerosis bacilli grow well on glycerine agar.

In addition to the white Staphylococcus, the Streptococcus may be present when
inflammation of the nasal duct exists.

The Streptococcus is at times responsible for a pseudo-membranous conjunctivi-
tis. The Staphylococcus is as a rule the cause of phlyctenular conjunctivitis.

382 EYE INFECTIONS

The Pneumococcus is a fairly common cause of serpiginous corneal ulcerati<
Active treatment is necessary.

It is now recognized as advisable to make an examination for the
Pneumococcus before performing operations on the eye as serious
results may follow if the Pneumococcus be present. It is the organism
frequently found in dacryocystitis and, in the case of traumatism, may
bring about panophthalmitis.

:

Corneal ulcerations are not apt to appear even with a pneumococcal conju
tivitis unless there be an injury of the epithelium.

The B. xerosis is possibly a harmless organism and must not be accepted as
plaining an infection unless other factors have been eliminated. The true diph-
theria bacillus, which the xerosis so much resembles, may cause a pseudomembran-
ous inflammation.

The B. pyocyaneus may cause severe purulent keratitis as well as conjunctivitis.
The pyocyaneus toxin appears to be a factor.

The Gonococcus and the Koch- Weeks bacillus are usually responsible
for the very acute cases of conjunctivitis. Both these organisms are
characteristically intracellular and are Gram negative.

Conjunctivitis in the course of epidemic cerebrospinal meningitis has been
found to be due to the Meningococcus.

The diplobacillus of Morax and Axenfeld is more common in chronic, rather
dry affections of the conjunctiva, chiefly involving the internal angle and showing
a morning accumulation of the secretion. The bacilli are found in twos, more
rarely in short chains. They are generally free but may be found in phagocytic
cells. They resemble Friedlander's bacillus morphologically but do not have
capsules.

In cases of ozena with involvement of the nasal ducts Friedlander's bacillus
may be found.

Even in cases without ozena, capsulated, Gram-negative bacilli of the Fried-
lander group have been frequently reported in conjunctival inflammation and in
dacryocystitis as well.

The nodules of the eye-brows give the most convenient area to take
material from in the diagnosis of leprosy, either the fluid expressed
after scraping or a piece of tissue cut into sections. Conjunctival ul-
ceration in leprosy may show abundant bacilli as is also true of corneal
ulceration.

Ordinarily it is impossible to find tubercle bacilli in tuberculous conjunctival
discharges.

The discharge from a tuberculous dacryocystitis may show them satisfactorily.
Animal inoculation is preferable in the diagnosis of ocular T. B. The Pneumo-
coccus is, however, the most important organism in dacryocystitis — rarely the B.
coli.

INFECTIONS OF THE OCCULAR REGION 383

In a gonorrhoeal ophthalmia the secretion is much more abundant and there is
an absence of contaminating organisms, the reverse of infection with the confusing
M . catarrhalis. As a matter of fact, large numbers of M . catarrhalis may be present
in the conjunctival secretion with only slight irritation being observable.

Wherry has reported two cases of ulcerative conjunctivitis with lymphadenitis
of cervical glands, fever and marked prostration, due to infection with B. tularense,
occurring in persons who had handled rabbits which had died of this plague-like
infection. The organism was first noted by McCoy in squirrels in California. The
symptoms and lesions in these animals are those of plague. Guinea-pigs succumb
after the cutaneous inoculation of material and show lesions markedly resembling
plague. The organism, however, will only grow on coagulated egg yolk, thus
differentiating it from B. pestis. McCoy has noted that the infection in squirrels
may be transmitted by fleas (Ceratophyllus acutus).

In keratomycosis the cause has been ascribed to Aspergillus fumigatus.

Certain fungi of the genus Microsporum have been thought to be
the cause of trachoma, as have also certain bacillary forms. One
should be very conservative about reporting fungi in smears or cul-
tures of external surfaces.

The larval stage of Tania solium (Cysticercus celluloses) has a predilection for
eye as well as brain. It is usually situated beneath the retina.

The question as to the nature of the so-called ophthalmic flukes is taken up under
trematodes. Echinococcus cysts have been reported in the orbit.

The adult Filaria loa tends at times to appear under the conjunctiva or in the
subcutaneous tissue of the eye-lids.

Fly larvae have been reported from the conjunctival sacs hi the helpless sick,
species of larval sarcophagids having been reported as invading the conjunctival
region in purulent ophthalmias.

Demodex may cause an obstinate blepharitis.

Prowazek has thought that certain fine dots within the cytoplasm of epithelial
cells, which stain best by Giemsa's method and which he considered protozoal in
nature, were the cause of trachoma. See Koch-Weeks bacillus and trachoma
bodies.

CHAPTER XXIV

DIAGNOSIS OF INFECTIONS OF THE NASAL AND A

CAVITIES

URAL

IN taking mateiial from the nasal cavities, for bacteriological ex-
amination, it is well to wash about the alae with sterile water and
then have the patient blow his nose on a piece of sterile gauze and take
the material for culture or smear from this. If the material is purulent
and located at some ulcerating spot, it is best to use a speculum, and
either touch the spot with a sterile swab or use a capillary bulb pipette
with a slight bend at the end.

Normally, we find only white staphylococcus colonies and colonies of short-chain
streptococci. The M. tetragenus, B. xerosis, and Hofmann's bacillus are also
occasionally found.

In some cases of ozena we may find an organism of the Friedlander type in pure
culture.

Biscuit-shaped diplococci, both Gram-negative and positive, are to be found
either normally or in cases of coryza. M , catarrhalis has probably been frequently
reported as the Meningococcus. Still, the Meningococcus has been found in the
nasal secretions of patients with cerebrospinal meningitis. B. influenza and the
Pneumococcus have also been frequently found in cultures from the nasal secretions.

Diphtheria involving the nasal cavity must always be kept in mind,
and in quarantine investigations the examination of the nasal secre-
tions culturally should be a part of the routine.

The tubercle bacillus may be found in nasal ulcerations; it is, however, only
present in exceedingly small numbers. On the other hand, one of the best diag-
nostic procedures in leprosy is to examine smears from nasal mucous membrane
for the B. hpra. In such ulcerations the bacilli are found in the greatest profusion.
Rarely glanders may cause ulcerations.

B. proteus is frequently responsible for the production of foul odors in nasal
discharges but does not seem to produce inflammatory conditions of the nasal
mucosa. It simply decomposes the discharges. Various fungi have been reported
from the nose, but in such a region the strictest conservatism in reporting should be
observed.

Recently sporozoa have been reported in a case of nasal polyp. (Rhinos poridium.)

So many degenerative changes in epithelial cells resemble protozoal forms that
such findings require ample confirmation.

384

INFECTIONS OF THE NASAL AND AURAL CAVITIES 385

The larval form of Linguatula rhinaria is a rare parasite of the nasal cavities;
it is not infrequent, however, in the nostrils of dogs.

Various fly larvae are far more common, and the "screw- worm,"
the larva of the Chrysomyia macellaria, is common in certain parts of
tropical America, and may by its burrowing effects cause fatal results.

The larvae of Sarcophaga have in particular been found in the nasal cavities of
children. Myriapods, while of very little importance elsewhere, have been reported
more than 30 times from the nasal fossae.

In a study of the bacteriology of otitis media, in 277 cases, Libman
and Celler found streptococci present alone in 81%, Streptococcus
mucosus in 10% and the Pneumococcus in 8%; Staphylococcus, B. pyo-
cyaneus and B. proteus have also been found. Mixed infections are
common.

Streptococci are the organisms which most often cause sinus thrombosis and brain
abscess. The influenza bacillus has been reported as a cause of acute otitis media.

Nonvirulent diphtheroid bacilli are not infrequently obtained in cultures from
ear discharges.

Other organisms which have been isolated from middle-ear or mastoid discharges
are B-. coli, M. catarr kalis, M. tetragenus and Friedlander's bacillus.

B. typhosus may be found in middle-ear discharges of persons who have had an
attack of typhoid fever.

The middle ear is normally free of bacteria, but in affections of the
throat, as with streptococci, pneumococci, and diphtheria bacilli,
these organisms may infect it by way of the Eustachian tube.

The moulds are of greater importance in affections of the external auditory canal
than the bacteria. The cerumen seems to make a good culture medium so that
various species of Aspergillus, Mucor, etc., develop and close the canal. These
infections are often introduced by the patient's finger. Various mites and fly larvae
have been reported from the ear.

The "screw worm," the larva of Chrysomyia macellaria, is the most common
cause of aural myiasis in tropical America. The fly deposits its eggs about aural
and nasal cavities of those with offensive discharges. The larvae develop and
cause intense pain and giddiness. Larvae of Sarcophaga, Calliphora and Anthomyia
have also been reported from the external auditory meatus. The tympanic mem-
brane may be perforated.

CHAPTER XXV
EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL

IN a preparation made from material taken by a sterile swab from
the region of the normal buccal and pharyngeal cavities and stained by
Gram's method we are struck by the variety of organisms present.
The fungi of thrush are best examined for in a preparation of membrane
mounted in 10% caustic potash solution. Manilla may be found in
sprue ulcerations about tongue or buccal mucosa. In examining for
buccal amoebae mount the purulent material from pyorrhoea in the

FIG. 104. — Vincent's angina. Spirochata vincenti. (Coplin.)

patient's saliva. Smears from about carious teeth often show the fusi-
form bacillus and delicate spirillum of Vincent as well as cocci.

Gram-positive and Gram-negative staphylococci are normally present, as are also
streptococci, pneumococci, leptothrix forms, and very probably yeasts and sarcinae
types with many Gram-negative bacilli. If pseudo-diphtheria organisms are
present, we have these showing a Gram-positive reaction. If this material is
smeared on agar plates and cultured at 37°C., we are struck by the fact that the
colonies on the plates may be exclusively staphylococcal and streptococcal.

Of course diphtheroids as well as diphtheria organisms grow well on ordinary agar
plates. For Meningococcus carriers a blood agar or starch agar plate is advisable.

It is very difficult, if not impossible, to distinguish a Pneumococcus colony from

386

EXAMINATION OF BUCCAL AND PHARYNGEAL MATERIAL 387

a Streptococcus one on a plate culture. The presence or absence, however, of the
Pneumococcus is distinctly shown in the Gram-stained smear, either by its lance-
shaped morphology or the presence of a capsule. It has been my experience that
smears from about 15% of normal individuals show capsulated pneumococci.

In diphtheria examinations we rely chiefly on the cultural findings on Loffler's
serum. Where the process is streptococcal or due to the organisms associated with
Vincent's angina, the immediate examination of a smear from the suspected spot
or area gives greater diagnostic information. The Streptococcus being so abundant
in cultures from normal throats, it is difficult to determine its significance in a cul-
ture; abundance of streptococci in a smear from an ulceration or bit of membrane,
however, is of etiological import. Streptococcal sore throats are often very toxic
and may be fatal — often milk borne. Use blood agar plates to differentiate hjemo-
lyzing and "viridans" types of streptococci.

By staining with Neisser's method it is possible to make an imme-
diate diagnosis of diphtheria from a smear from a piece of membrane
in about 25% of cases. It is well, however, to always culture such
material. The toluidin blue stain of Ponder is the best stain for
diphtheria.

Material from the throat is ordinarily best obtained with a sterile copper-wire
cotton-pledget swab. The platinum loop usually bends too easily. A sterile for-
ceps may be more convenient for obtaining particles of membrane. It is believed
that ulcerative conditions of the throat,' associated with the presence of the large
fusiform bacillus and delicate spirillum, which make the picture of Vincent's angina,
are more common than is usually so considered.

In Giemsa-stained smears from the dirty membrane covering the ulcerated area
of Vincent's angina there are usually two types of the fusiform bacillus to be seen;
one rather slender, pale blue with maroon dots at either end, the other rather thicker
and of a uniform maroon staining. The spirilla are from 10 to 18 microns long and
the fusiform bacilli from 5 to 7 microns.

As a rule, only cultures on Loffler serum are made and very rarely direct smears.
If a smear were always made and stained by Gram's method (with a contrast stain
of dilute carbol fuchsin) at the same time the culture was made, it is probable
that much information of value would be obtained.

The B. fusiformis is an anaerobe which gives a fetid odor but cul-
turally has no distinct characteristics. The spirillum has not been cul-
tivated. It has been thought that the bacillus and spirillum are
different stages of the same organism.

At times aggregations of the fusiform bacillus give the appearance of branching so
characteristic of diphtheria organisms. Being Gram-negative, however, the differ-
entiation is easily made— the B. diphtheria being Gram-positive. Again the attenu-
ated ends of the fusiform bacillus are diagnostic.

It is usually stated that the fusiform bacillus is nonmotile. By mounting material
in saliva I have noted a sluggish, but distinct motility. The fusiform bacillus

388 VINCENT'S ANGINA

and spirillum are often associated with cocci and amoebae in pus from dental caries
or pyorrhoea and I mount such material in the patient's saliva to obtain motility
in the amoebae. The fusiform bacillus is not markedly Gram-negative.

The culturing of material from the nasopharyngeal region of contacts as well as
patient is very important in outbreaks of cerebrospinal fever. Use a bent wire
applicator with sterile cotton tip and pass it to the nasopharynx avoiding the uvula.
Inoculate tubes of serum or blood agar immediately.

Direct smears are the procedure of choice in streptococcal and
pneumococcal anginas as well as in Vincent's angina.

Unless very familiar with the morphology of Treponema pallidum and using
dark field or Fontana's staining procedure, we should be very conservative in report-
ing such an organism from suspected syphilitic ulcerations of the throat.

We now know that we have treponemata in the buccal cavity similar to T. pal-
lidum so that even with the dark field illumination I would base a diagnosis on
the clinical and Wassermann reactions rather than morphologically.

The thrush fungus (Endomyces albicans) may be easily demonstrated in a Gram-
stained specimen as violet mycelial structures.

Yeasts due to food particles are not infrequently observed in smears and cultures
from the mouth.

Actinomycosis may develop about a carious tooth and the finding of
the ray fungus in the granules from the pus may give the diagnosis.

Amoebae and flagellates have been reported from the mouth. Also in the re-
markable disease "halzoun," flukes have been found to be the cause of the asphyxia.

In the tropics, round worms (A scar is] may be vomited up and, lodging in the
pharynx, may have to be extracted.

During the campaign of Napoleon in Egypt many cases of leech involvement of
the nasal and buccal cavities were noted. The parasite was the Limnatis nilotica
which gained access to the upper pharynx through drinking water from springs and
pools. Many such cases continue to be reported from the Mediterranean basin.

CHAPTER XXVI
EXAMINATION OF SPUTUM

FREQUENTLY the material submitted for examination as sputum is
simply buccal or pharyngeal secretion, or more probably secretion
from the nasopharynx, which has been secured by hawking. It should
always be insisted upon that the sputum be raised by a true pulmonary
coughing act, and not expelled with the hacking cough so frequently
associated with an elongated uvula. When there is an effort to deceive,
some information may be obtained from the watery, stringy, mucoid
character of the buccopharyngeal material and also from the presence
of mosaic-like groups of flat epithelial cells (often packed with bacteria).
The pulmonary secretion is either frothy mucus or mucopurulent
material, and if the cells are alveolar they greatly resemble the plasma
cells. At times these cells may contain blood pigment granules (heart-
disease cells).

In the microscopic examination a small, cheesy particle, the size of a pin head,
should be selected. This should be flattened out in a thin layer between the slide
and cover-glass and should be examined for elastic tissue, heart-disease cells, eggs
of animal parasites, amoebae, and fungi. Echinococcus booklets, Curschman spirals
besprinkled with Charcot-Leyden crystals, and haematoidin and fatty acid crystals
may also be observed.

Curschman spirals indicate bronchial as against cardiac or uremic asthma. Char-
cot-Leyden crystals have no special significance, except in certain tropical diseases
when these crystals often are present in paragonomiasis sputum and in the pus
of amcebic liver abscesses discharging by way of the lungs.

It may facilitate the examination of the sputum for elastic tissue
and actinomycosis and other fungi to add 10% sodium hydrate to the
preparation.

To make smears for staining, the sputum should be poured on a flat surface,
preferably a Petri dish, and a bit of mucopurulent material selected with forceps. A
dark back-ground facilitates picking out the particle. A toothpick is well adapted
to smearing out such material on a slide. After using the toothpick it can be burned.
When dry, the smear is best fixed by pouring a few drops of alcohol on the slide,
allowing this to run over the surface, and then, after dashing off the excess of alcohol,
to ignite that remaining on the film in the flame and allow to burn out.

389

390

ANTIFORMIN

merit

A mark with a grease pencil, about ^ inch from the end, gives a convenient
surface to hold with the forceps and also prevents the stain subsequently used from
running over the entire surface. A piece of glass tubing about 1 2 inches long bent
into a narrow V shape makes a very satisfactory rest for the slide in staining and is
convenient for the steaming of staining solution over the flame.

Sputum should as a routine measure be stained by the Ziehl-Neelson method and
by Gram's method.

In examining for tubercle bacilli it may be necessary to employ some method for
concentrating the bacterial content of the sputum prior to making the smear. A
very satisfactory method is that of Miihlhauser-Czaplewski. Shake up the sputum
with four to eight times its volume of %% solution of sodium hydrate in a stoppered
bottle. When the mixture has become a smooth, mucilaginous-looking fluid, add a
few drops of phenolphthalein solution and bring the pink mixture to a boil.

Then add drop by drop a 2% solution of acetic acid, stirring constantly, until
the pink color is just discharged. If the least excess of acid is added over that just
sufficient to cause the pink color to disappear, mucin will be precipitated. Now pour
this mixture into a centrifuge tube and smear the sediment on a slide and stain for
tubercle bacilli.

Antifonnin. — Tubercle bacilli usually occur nested in clumps of
sputum. Therefore, when few in number it is only by chance that they
may be found. Concentration methods aim to dissolve these clumps
of sputum and collect, free from mucus, whatever bacilli may be
present. There are many concentration methods for sputum. One
of these has been given above. Uhlenhuth's method has some
advantages over others in the solvent used: i. It breaks up the
sputum very rapidly; 2. it immediately dissolves all organisms except
acid-fast ones; 3. applied in not too concentrated form and for not
too long a time, tubercle bacilli are not killed, so that by washing
the sediment carefully by several dilutions and centrifugings we have
in the sediment viable tubercle bacilli which we may attempt to
cultivate upon Dorsett's or other suitable media with the reasonable
hope that contaminations will not choke them out or prematurely
kill the inoculated guinea-pig; 4. it has less effect upon the staining
properties of tubercle bacilli than any other material used in
concentration methods. PetrofTs method is probably better.

To make this solvent (antiformin) take double the quantity of chlorinated lime
and sodium carbonate required by the U. S. Pharmacopoeia and prepare according
to U. S. P. directions. To the finished liquor sodae chlorinatae (Labarraque's solu-
tion) add 7^% of sodium hydrate.

The Liquor sodae chlorinatae 'of the Br. P. is slightly stronger and some English
authorities recommend a mixture of equal parts of this Labarraque's solution and
15% sodium hydrate solution. As a rule i part of antiformin to 5 parts of sputum
is sufficient. Very tenacious sputum may require i part to 4 parts of sputum.

EXAMINATION OF SPUTUM 391

If more antiformin is used the specific gravity is too much increased and the bacilli
are damaged. The fluidification is hastened at incubator temperature.

To 5 parts of sputum add i part of antiformin, shake well and place in incu-
bator for one hour. To 10 c.c. of the homogeneous mixture add 1.5 c.c. of a
solution made up of i part chloroform and 9 parts alcohol. Shake violently and
centrifuge for fifteen minutes. Mix the sediment with egg albumin, smear out and
stain.

When it is desired to culture the tubercle bacilli mix 20 c.c. of sputum with 65 c.c.
sterile water and add 15 c.c. antiformin. Stir the mixture with a glass rod. After
thirty minutes to two hours we should have a homogeneous mixture. Centrifuge
for fifteen minutes or longer, wash the sediment twice with sterile salt solution and
smear out the well- washed sediment over serum or glycerine egg slants. The tubes
should be covered with black paper and the plugs paraffined. It must be remem-
bered that for culturing tubercle bacilli we must protect the growth from sunlight
as this will kill the organism. If fluid culture media are inoculated the transferred
material should be deposited on the surface. Should the particle sink growth will
not occur.

Sputum smears stained by some Romanowsky method or by the
haematoxylin-eosin stain are best adapted for the study of various
cells, and in particular of the eosinophile cells so characteristic of
bronchial asthma. In sputum from cancer of the lungs the large
vacuolated cells may be found.

When examining the sputum of the bronchopneumonia of influenza the formol
fuchsin gives the best results. The influenza bacilli are found in little masses, fre-
quently grouped about small collections of M. tetragenus. The cocci stain a rich
purplish-red, while the small influenza bacilli take on a light pink color.

A greenish yellow, nummular sputum, often profuse, is frequently noted in in-
fluenza.

T. B. sputum showing a mixed infection with streptococci or pneumococci or with
the influenza bacillus makes for a bad prognosis. M. tetragenus, which often is
present when cavities exist, does not seem to be so unfavorable prognostically.

Red cells show up well in specimens stained by the Romanowsky method; if
rouleaux formation is marked, it may indicate pulmonary infarction.

In culturing sputum a mucopurulent mass should be washed in
sterile water and should then be dropped into a tube of sterile bouillon.
With a sterile swab it should be emulsified and successive streaks made
along the surface of an agar, blood agar or glycerine agar plate. In
obtaining cultures from influenza sputum, first smear the material
thoroughly over a blood-serum slant; then inoculate, by thorough
smearing over the surface of successive blood-streaked agar slants, the
material on the surface of the blood-serum slant. The platinum loop
should be transferred from one slant to another without recharging.
The influenza bacillus seems to grow better if the blood-streaked agar

392 ALBUMEN IN SPUTUM

11 that

slants are prepared just before inoculating with the sputum. All
is necessary is to sterilize an ear, puncture and take up the exuding blood
with a large loop and smear over the agar slant. Cultures for tubercle
bacilli are impracticable except with antiformin or by PetrofTs method.
This latter is most satisfactory. A guinea-pig should be inoculated.

The blood-stained watery sputum of plague pneumonia should be cultured on
plates of plain agar and 3% salt agar at the same time. An ordinary smear stained
with carbol thionin, however, practically makes a diagnosis. Be sure to inoculate
a guinea-pig cutaneously.

Pneumococci, M. catarrhalis, and Friedlander's bacillus in sputum are best
demonstrated by Gram's method of staining.

The distinct capsule staining of the pneumococci in a Gram preparation of
sputum from a suspected case of pneumonia is of value in diagnosis.

The finding of the ray fungus (D. bovis) in sputum gives the diagnosis of actino-
mycosis. Streptothrix infections of lungs have been confused with tuberculosis.

Moulds, especially aspergilli, may be found in sputum. Species of Mucort
Cryptococcus, and Endomyces have also been reported.

Amoebae from liver abscess rupturing into the lung may be found.
Very important pulmonary infections are those with Paragonimus
westermanii. This is recognized by the presence of operculated eggs
in the sputum.

A fluke, F. giganlea, was once found in sputum.

Hydatid cysts, either of the lung or of the liver, rupturing into the lung, may be
recognized by the presence of echinococcus booklets. The material is bile-stained
if from the liver. Dutcher has reported filarial embryos from sputum.

Strongylus apri has been reported once from the lungs and embryos might be
found in the sputum. In pulmonary bilharziosis Schistosoma egggs may be found in
the sputum.

The test for ALBUMEN IN THE SPUTUM is of value in the diagnosis of pulmonary
tuberculosis.

About 10 c.c. of fresh sputum as pure as possible from saliva is mixed with an
equal quantity of water and 2 c.c. of a 3% solution of acetic acid to remove mucin.
After filtering the filtrate is tested for albumin. The test is obtained also in
pneumonia and pleurisy with effusion.

CHAPTER XXVII
THE URINE

MATERIAL -for staining is best obtained by centrifuging the urine,
then pouring off the supernatant urine, then draining the mouth of
the centrifuge tube against a piece of filter-paper so that we have only
the pus sediment to finally remove with a capillary bulb pipette or
toothpick stuck in a urine sediment pipette and make smears.

I always take up the material with the centrifuge tube in a slanting position
following the draining off of the supernatant urine to avoid urine admixture in
the smear as such makes staining less satisfactory. The Gram staining is most satis-
factory, counterstaining with bismarck brown.

The addition of a loopful of egg albumen or blood serum to about twice that
amount of urinary sediment gives better results. (See under Staining Methods.)

In pathological urine there is enough albumin to fix the smear.

The smear may be stained after fixing by heat with Gram's stain, T. B. stain,
or haematoxylin and eosin. The latter is the best for the staining of epithelial cells
and animal parasites; the Gram method for bacteria.

It is frequently difficult to distinguish the spores of moulds from red blood-cells
except by measurement and staining reactions. Spores of moulds rarely exceed
five microns.

Of the greatest value is the finding of phagocytized bacteria in the
pus cells of the Gram-stained smear. These indicate the causative
organism which show beautifully in the beginning of pyelitis infections.
To examine for epithelial cells I make a vaseline streak across a slide
about % inch from the center. A drop of the sediment is deposited
on the slide which may then be examined unstained with the % inch
objective and then a drop of Gram's iodine solution is added. One
edge of a square cover-glass is rubbed into the vaseline line and allowed
to drop on the fluid preparation. Currents are avoided and the cells
stain up beautifully.

It is difficult to determine the presence of blood in urine in higher dilution than
i to 300 with the spectroscope. The ordinary occult blood test will show it in much
higher dilution.

To secure urine for bacteriological examination catheterization is rarely necessary
in men — in the case of women it is the proper method.

393

394

CULTUEING URINE

icn the

Authorities generally insist upon a catheterized specimen in all cases when
urine is to be cultured. As a matter of fact when there is a bacterial infection the
specific organism is in such predominant numbers that it is easily distinguished
from a possible contaminator. Of course should one culture the urine in a tube of
bouillon, before plating out, a contamination might overgrow the causative organism,
but one should always plate directly from urine which has just been passed. The
smear stained by Gram's also checks up, particularly if certain bacteria are found
phagocytized in pus cells. The man who follows the clinical side as well as the labora-
tory one is rarely confused by an occasional contaminating organism on a plate
made from urine or blood. Of course the problem is more difficult with urine,
but when culturing of urine is a routine procedure the worker soon knows the organ-
isms likely to be encountered in urine of women as well as that of men. As a matter

Oxyhemoglobin

Methemoglobin

Reduced
Hemoglobin

CO Hemoglobin

FIG. 105. — Principal Blood Spectra. (Da Costa.}

of fact I rarely find colonies on plates made from the urine of normal men, the only
precautions taken being those noted below. I now use blood agar plates as routine
plating media.

The glans penis and meatus should be thoroughly washed with soap and water,
after which dilute alcohol (70%) should be used. The greater part of the urine
first passed should be rejected and only the last portion passed should be caught
in a sterile receptacle. A drop of this urine may be either streaked over the
surface of an agar, blood agar, or a lactose litmus agar plate, or so treated after
being first diluted in a tube of sterile bouillon.

The lactose litmus agar medium is very useful in distinguishing
typhoid or paratyphoid colonies (blue) from colon, and Streptococcus

THE URINE 295

or Staphylococcus colonies (pink). The urine may be added to tubes
of melted agar and then poured.

The most satisfactory procedure is to deposit one drop on a poured plate and five
drops on a second plate. The surface is smeared over with a bent glass rod first
smearing out the single drop and then going to the second plate without a second
sterilization. Neutral glycerine agar or blood agar is desirable for such organisms
as pneumococci or streptococci and, for the Gonococcus, Thalman's medium smeared
over with a few drops of human serum or Vedder's starch agar.

Cystitis from a colon infection gives an acid urine; that caused by Proteus vulgar is
an alkaline urine.

The old designation B. termo so often employed in connection with the bacteri-
ology of the urine in older works applied to the proteus group and M . urea to ordi-
nary staphylococci.

The bacillus of typhoid and the micrococcus of Malta fever are also
found in the urine. This elimination in urine of bacilli by typhoid
carriers is of great importance in the spread of the disease.

While the smegma bacillus in urine may be differentiated from the tubercle
bacillus by the former losing its red color, by prolonged decolorization with acid
alcohol, yet it is chiefly by the subcutaneous inoculation of the guinea-pig that we
should diagnose genito-urinary tuberculosis. Inject the sediment after centrifuging.

The method recommended by Gasis which depends on the alkali fast properties
of the T. B. has not given me satisfactory results.

Smegma bacilli are not disintegrated by antiformin as are other bacilli than the
tubercle one, so that treatment of urinary sediments with antiformin for finding
tubercle bacilli does not differentiate those of smegma.

Gonococci are reported from Gram-stained smears.

To culture Gonococcus material the transfer to culture media should be made
almost immediately after obtaining the material from the patient. M . catarrhalis
is a rare finding.

Staphylococcus and Streptococcus infections about the throat as well
as such infections in heart or joint may show the presence of the causa-
tive organisms in the urine. At times bacterial infections of the kidney
may give symptoms of renal stone.

As it is much easier to culture urine than blood a bacteriological examination of
the urine may give us the desired information and the organism for the autogenous
vaccine. Salt mouth bottles with cotton plugs, when sterilized, make cheap and
satisfactory containers, The urine should be plated out as soon as possible after
its passage. As a rule when organisms are present in the urine they are in such
numbers that the question of contamination rarely arises.

Yeasts and moulds frequently contaminate urine, especially diabetic urine, after
it has been passed. Amoebae and flagellates (Trichomonas vaginalis in females)
may be found in urine.

396 URINARY SEDIMENTS

irlincrc •

Eggs of Schistosoma hcematobium (bilharziosis) are important diagnostic findings;
these are terminal-spined. Those of rectal bilharziosis are, as a rule, lateral-spined.

In chylous urine the filarial embryos may be found. This exami-
nation is facilitated by centrifugalization.

The eggs of the E. gigas may be recognized in urinary sediment by their pitted
appearance.

The vinegar eel may be found in the urine of females who have used vaginal
douches of vinegar.

Echinococcus booklets, scolices, or laminated membrane have been found in the
urine.

The larval dibothriocephalid, Sparganum mansoni, has been reported three times
in urine (urethra).

Oxyuris from the vagina may be found in urine.

Various mites may be found in urinary sediment as the result of lack of care
the washing of the receptacle and are entirely accidental.

ics

:

Unless having the characteristics of the itch mite and in a persoi
showing scabies lesions about the genital organs the diagnosis of the
mite as A. scabiei should not be made.

Crystals of biliverdin may be found in the urinary sediment in marked jaundice.
They somewhat resemble crystals of tyrosin but are brownish in color while those
of tyrosin are black. Furthermore, it is excessively rare to find crystals of leucin
and tyrosin in the urinary sediments, and in such diseases as acute yellow atrophy of
the liver, the urine should be concentrated to one-tenth its volume and the residue
treated with alcohol. The tyrosin crystalline sheaves and the leucin striated
globules crystallize out from the alcohol.

URINARY SEDIMENTS

Turbidity of the urine is most often due either to bacterial contamination, amo
phous urates (sedimentum lateritium) or phosphates.

Urates go into solution upon heating and phosphates upon the addition of a few
drops of acetic acid.

In turbidity due to bacteria contaminating the urine subsequent
to its passage it is best to call for another sample.

To preserve urinary sediments formalin is the best for casts and epithelial ce
while for general use one may employ a piece of camphor or the addition of o
volume of saturated borax solution to four volumes of urine.

Chloroform does not answer for sediments as it does for urine to be examined
chemically. To take up a sediment insert a pipette to the bottom of the tube with
the opposite opening closed by a finger, then tease the sediment into the pipet
opening in the centrifuge tube, by manipulating the fingers.

THE URINE

397

In a urine of acid reaction we may find the following unorganized sediment:

I. Amorphous sodium or potassium urates. Usually yellowish red. Heat and
alkali bring about solution.

II. Uric acid. Whetstone crystals of yellowish-red color. Soluble in alkalis

FIG. n;o6. — Deposit in acid fermentation, a. Fungus; b, amorphous sodium urate;
c, uric acid; d, calcium oxalate.

but not by heat. Abundant sediment of uric acid crystals may be due to too great
concentration or too great acidity of the urine rather than to the so-called uric
acid diathesis.

-b

FIG. 107. — Deposit in ammoniacal fermentation, a, Acid ammonium urate, d;
ammonium magnesium phosphate; c, bacteria.

III. Calcium oxalate. Octahedral crystals or dumb-bell shapes which are highly
refractile. Often due to diet (asparagus, tomatoes, spinach, rhubarb, etc.).

IV. Cystin occurs in six-sided crystals which are soluble in ammonia.

398

EPITHELIAL CELLS IN URINE

In a urine of alkaline reaction we may expect:

I. Triple phosphates (NH4 MgPO4). Usually in coffin-lid or fern-like form.
Easily soluble in acetic acid.

II. Calcium phosphate and calcium carbonate which effervesce on the addition
of acid.

III. Ammonium urate. These show as the thorn-apple structures.

The presence of ammonium urate, particularly if with triple phosphates, denotes
bacterial decomposition within the genito-urinary tract provided the urine is just
passed. Pus cells derived from the site of inflammation should be present also.
While certain bacteria might possibly bring on chemical changes without giving rise
to inflammation yet such a possibility is so rare as to be negligible. In the presence
of amorphous phosphates one should always think of exogenous sources as vegetable
diet or withdrawal of proteid food before thinking of disordered metabolism.

FIG. 108. — Epithelium from different areas of the urinary tract, a, Leukocyte
(for comparison); b, renal cells; c, superficial pelvic cells; d, deep pelvic cells; e,
cells from calices; /, cells from ureter; g, g, g, g, g, squamous epithelium from the
bladder; h, h, neck-of-bladder cells; i, epithelium from pros ta tic urethra; k, urethral
cells; /, /, scaly epithelium; m, m', cells from seminal passages; n, compound granule
cells; o, fatty renal cell. (Ogden.)

Organized Sediment. — An occasional leukocyte may be found in the urine of
healthy people. Any abundance of leukocytes indicates inflammation of genito-
urinary tract. Some workers count the pus cells in urine by the same technic used
for the leukocyte count of the blood. A urine having 100,000 pus cells per c.mm.
will give as a result about 0.1% albumin.

Leukocytes are found in abundance at times in the urine of women without
pathological significance. Red blood cells usually show as pale doubly ringed
bodies. They appear in inflammations, particularly stone or schistosome infection,
also with neoplasms or in chronic passive congestion. They may be found in
conditions where toxins are being eliminated through the kidneys, as in tubercu-

THE URINE

399

losis. The menstrual period of women must be kept in mind in the examination
of urine sediments.

Epithelial Cells. — For morphology of cells from different locations see illustration.
It is almost impossible to state positively the origin in the genito-urinary tract of
certain cells. Very trustworthy evidence however is finding of epithelial cells on
casts or the so-called compound granule cells (fatty degenerated renal epithelium).
Sheets of more or less small round or caudate epithelial cells are rather significant of
pyelitis. Vaginal epithelium resembles that gotten from scraping the buccal
mucosa.

Bladder epithelium resembles vaginal but is of smaller size, and ureteral is like
that of the pelvis of the kidney but smaller. Cells from the region of the prostate
are very refractile with a distinct nucleus and are oval rather than round.

FIG. 109.— Fatty and waxy casts, a, Fatty casts; b, waxy casts. (Greene.')

Casts.— Of casts we have (i) hyaline, narrow and homogenous.
They do not prove nephritis. (2) Epithelial casts. Usually indicative
of nephritis but very slight inflammatory processes can cause them.
(3) Blood casts. (4) Granular casts. If coarse granules rather sig-
nificant of chronic nephritis. Finely granular casts do not seem to
have any more significance than hyaline ones. As a matter of fact
under dark ground illumination hyaline casts show a granular structure.
(5) Waxy casts are highly refractile, show fissuring of margins and were
formerly considered of serious prognostic import (chronic nephritis),

40O

STARCHES AND FIBRES IN URINE

but the present view is that they are only ordinary casts which have
been retained in the renal tubules for a long time. Even amyloid
kidney does not produce any distinctive cast.

Cylindroids are drawn-out bodies showing tapering ends, irregularity of diameter
and longitudinal striations.

It will be found that a ^ in. objective gives almost all the information required
as to casts. It is quicker and gives more positive information.

Mounting a sediment in Gram's solution or tinging it with the merest trace of
neutral red is of much assistance.

FIG. no. — Fibres, starch granules, etc., which may be found in urine sediment.
No. 12 gives appearance under microscope of scratches on old used glass slides.
No. 1 5 (a) , Tyroglyphus longior, a mite. No. 1 5 (b) , Trichomonas vaginalis. No. 1 6 (a) ,
Egg of Eustrongylus; (6), Echinococcus hooklets', (c), Schislosoma egg; and (d),
Filaria bancrofti embryo.

Starches and Fibres. — In examining urinary sediments it is important to be fa-
miliar with the various textile fibres and starch grains which are so frequently present,
the fibres coming from the clothing and the starch grains from dusting powders.
Wool fibre fragments show bark or scale-like imbrications and are round. Cotton
fibres are flattened and twisted, while linen ones show a striated flattened fibre with
frayed segments as of a cane stalk. Silk shows a glass-like tube with mashed in ends.

Corn and rice grains are the most common of the starch grains and their nature is
immediately disclosed by their blue color when mounted in iodine.

THE URINE 40I

Haematuria.— By this we mean the presence of red blood cells in
the urine, and the condition is of ten recognizable only by microscopic
or occult blood examinations. It is better to separate this condition
from haemoglobinuria. Origin may be renal, cystic, urethral, ureteral
and secondary to disease or traumatism. It may also result from
affections of structures adjacent to the genito-urinary apparatus,
especially ulcerations of the large intestines (amoebic), or from disease
of the female generative apparatus as uterus, vagina or tubes.

In certain general diseases, as smallpox, purpura, and leukemia one may expect
hagmaturia. In the tropics it is a finding in yellow fever (asthenic stage) and in
plague as well as in bilharziasis and haematochyluria of filariasis.

Haemorrhage from the kidneys may arise from malignant growths, especially
hypernephroma, or from benign ones, as papilloma of the pelvis.

Haematuria occurs not only in acute nephritis but in some chronic
cases. Chronic passive congestion is associated with red cells in the
urine and renal infarctions from endocarditis may also bring it about.

Stone in the pelvis of the kidney is an important cause. In the bladder we have
as chief causes (i) tumors, malignant or innocent, and (2) calculus. In tumors
the bleeding is not markedly controlled by rest as is that from stone.

In the urethra gonorrhoeal inflammation, especially when near the neck of the
bladder, and traumatism may be associated with hsematuria.

Haemoglobinuria. — The two diseases one always thinks of in con-
nection with haemoglobinuria are blackwater fever and paroxysmal
haemoglobinuria. It is discussed under tests for transfusion.

Bacteriuria. — Bacterial infections of the genito-urinary tract are
associated with more or less pyuria. Kidney infections are now
recognized as most often from the blood stream rather than from
extension from portions of the tract lower down.

The most important hsematogenous bacterial infections of the kidney are, in
order of frequency, colon bacillus, staphylococci, streptococci, gonococcus, proteus,
typhoid and paratyphoid. Renal tuberculosis is generally haematogenous rather
than an ascending infection. Very important is the differentiation of the pyuria
of cystitis and pyelitis. Of course bladder irritation will follow pyelitis.

The use of two sedimentation glasses will differentiate pus from the urethra from
that from bladder and pelvis of kidney. If the urine in the first glass alone is turbid
it shows urethral pus. With cloudiness of the contents of the second glass the
problem is more difficult because it is almost impossible to differentiate a bladder
involvement from a renal one by microscopic examination. Cystoscopy is necessary.
Of course, some authorities attach importance to the character of pelvis epithelium,
others to the acid urine of pyelitis and the alkaline one of cystitis. Again it is
26

402 AMBARD 's INDEX OF UREA EXCRETION

often noted that the albumin content of the urine of pyelitis is far greater than that
from cystitis. This is true for pyelonephritis but does not hold for simple pyelitis.

Pyelitis, usually a colon infection, must always be thought of in
vague disorders of children and pregnant women.

Renal stone and renal tuberculosis often give similar symptoms but the X-ray
and animal inoculation test should differentiate.

DETERMINATION OF EFFICIENCY OF RENAL FUNCTIONING

At present we are paying great attention to laboratory tests which
give us an idea of the activity of nitrogen metabolism and efficiency
of the renal functions. Probably the most reliable single test is the
phenolsulphonephthalein or "red" test. This is given in the appendix.
For alveolar CO2 content see Acidosis.

Determination of the ammonia output in the urine is also of value in conditions
of acidosis. In acidosis connected with diabetes we expect a great increase in the
urinary ammonia to neutralize acetone bodies as is also true of degenerations invol-
ving the parenchymatous liver cells, when the urea function is interfered with.
In the acidosis of chronic nephritis, however, we may have a deficiency in the ammo-
nia output in the urine. Simple tests for this determination will be found in the
appendix.

Great attention is being given to the nonprotein nitrogen of the blood as well
as to blood urea. See appendix.

The study of nitrogen metabolism is best undertaken with a patient
on a known nitrogen value diet and the most accurate determination
is along the lines of the Ambard index of urea excretion.

Ambard Index. — McLean has worked out an index of urea excretion based on
the Ambard variables of (i) concentration of urea in the blood. (2) Concentration
of urea in the urine. (3) Rate of urinary excretion and (4) weight of the individual.
McLean's formula is as follows:

_ £.

= Index of urea excretion.

D = Grams urea excreted in twenty-four hours.

C = Grams urea per liter urine.
Ur = Grams urea per liter blood.
Wt = Eody weight, individual in kilograms.

One hundred is accepted as a typical normal finding, and findings down to 80
as within normal limits. In deficiency of renal function the index falls below 50
and in cases of marked deficiency may fall below 10, and in terminal stages
index may approximate i. Such cases practically show an absence or only a trace
phenolsulphonephthalein excretion.

sthe

..

THE URINE 403

For determination of urea excretion the patient drinks 200 c.c. water and one-
half hour later empties his bladder. Commencing at this we wait seventy-two
minutes (one-twentieth of twenty-four hours) and then collect the urine for urea
determination.

The blood for urea determination should be taken midway in the period of col-
lection of the urine — thirty-six minutes after the bladder is voided.

Salt Retention. — As a test for salt retention Schlayer advises giving
10 grams of NaCl to a patient having a salt equilibrium as the result
of a known diet continued over several days. This added salt should
be eliminated in the urine within twenty-four hours or certainly by
forty-eight hours. Monakow in a similar way gives 20 grams of urea
at one dose and this added urea should be eliminated in twenty-four
to forty-eight hours to show normal nitrogenous output.

A case of nephritis may show a good urea elimination but salt retention.

Diminished capacity to eliminate salt is a rather constant finding in all types of
chronic nephritis.

Frothingham considers elimination of 8.5 grams of the 10 grams administered as
normal, above 4.5 grams as slight retention and below 4.5 grams as marked retention.

Lactose Excretion. — Intravenous injection of 20 grams lactose in 20
c.c. distilled water. The solution should be pasteurized at 8o°C. for
three hours on three successive days. The urine is collected at one-
to two-hour intervals and tested with Nylander's test for sugar until
you get negative reaction. Normally all lactose is excreted in four
or five hours.

404

THE URINE IN RENAL DISEASE

JTJ i

>>e UJ

8^.2 o c §

§

a«S« ^
•38 g.2.pO,

p'sllJJ

II

w£

i!
fii

i*

f
us

eacti
No te

3-a

"rt o

eaction of
very faint
Tenesmus.

S3 . •

o),G rt C

ill!

|M

i!

Abundant.
casts inclu
waxy. R
Much fatt

§S
^°

.9

edimentum lateriti
occasional hyaline C£
Red and white bl<
cells. Renal epithel
exceptional.

bundance of pus
Caudate epitheliu
en in clusters. At
red blood-cells.

0 C

s

1

CHAPTER XXVIII
THE FAECES

IT is advisable to examine a stool macroscopically before taking up
the microscopical examination. The mucus shreds or casts of the
bowel in mucous colitis or membranous enteritis may give the diag-
nosis of obscure abdominal pain. Pus in stools may often be noted
without the aid of the microscope.

The normal stool is sausage shaped and soft. Neither the special form of scybal-
ous masses called sheep pellets nor the pencil-like nor the tape-like excrement prove
the existence of stricture of the intestinal lumen although suggestive of such a con-
dition. The mucus of bacillary dysentery is opaque and grayish from the great
number of pus and phagocytic cells. It is well to remember that Charcot Leyden
crystals, which are practically always absent from bacillary dysentery stools, are
not infrequent findings in the amoebae containing stools; of course, these crystals
appear in other intestinal parasite infections.

In obstruction of the common bile duct we have acholic, whitish, foul-smelling
stools. If the putty color be due to bacterial change exposure to the air will restore
the brownish tinge.

Sprue stools are white-wash to putty colored, pultaceous, and filled with air
bubbles. The amount is excessive.

Fatty stools are best examined microscopically.

As so many solid masses resemble gallstones it is well to dissolve the suspected
mass in hot alcohol and examine for cholesterin crystals upon evaporation of the
alcohol.

If the faecal examination is to be made for the diagnosis of amoebae,
in a case where the characteristic mucus stools are not present, or to
verify the existence of flagellates, it is best to give a dose of salts early
in the morning and examine the liquid stools which follow such treat-
ment. This treatment is satisfactory for examination for intestinal
parasites or ova.

A very practical way of obtaining amoebae is to pass a rectal tube or a piece of
drainage tube with fenestrations into the bowel, and amoebae may be found in the
mucus filling the perforations in the tube. Walker advises against the use of salts
in examinations for amoebae.

If the purpose of the examination is to determine the digestive power
of the alimentary tract for proteids, carbohydrates, or fats, it is best
to use a test diet, as that of Schmidt and Strasburger.

405

406

SCHMIDT TEST DIET

TTiT^if*

Prior to using this test diet, one should familiarize himself with the macroscopic
and microscopic appearances resulting from such a diet in a normal person; informa-
tion is then at hand to judge of variations from the normal. The examination of
the faeces of persons, on ordinary and specifically undetermined articles of diet, is
very unsatisfactory when the state of digestion of muscle fibers and the question of
fat digestion are at issue.

In examining the faeces of the normal person and likewise with the patient, wait
until the second or third day so that the faeces of previous diets may have passed out.
A charcoal powder taken before commencing the diet serves as an indicator.

Diet: breakfast, 7 A. M., bowl of oatmeal gruel (40 grams oatmeal, 10 grams
butter, 200 c.c. milk, 300 c.c. water). Also one very soft-boiled egg (one minute)
and 50 grams zwieback. In the forenoon, 500 c.c. of milk.

For dinner, 2 o'clock, chopped beef broiled very rare (125 grams with 20 grams

FIG. in. — Microscopical constituents of faeces, (v. Jaksch.) a, Muscle fibres;
6, connective tissue; c, epithelium; d, leukocytes; e, spiral cells; /, g, h, i, various
vegetable cells; k, "triple phosphate" crystals; /, woody vegetable cells; the whole
interspersed with innumerable microorganisms of various kinds.

butter poured over it). Also a potato puree (200 grams mashed potato, 50 grams
milk, 10 grams butter). Also J^ liter of milk and 50 grams zwieback.

For supper, 7 o'clock, the same articles as for breakfast.

This detailed diet may be varied to suit circumstances as regards interchanging
meals. Furthermore, the milk may be taken in the form of tea or cocoa or cooked
with the other food. Even a small amount of wine may be permitted. The diet
taken, however, should absolutely conform to the following requirements: i. the
taking of Y± pound chopped beef, a portion of which should be half raw; 2. the milk
taken should amount to about a quart; 3. about 4 ounces of bread or toast and from
4 to 8 ounces of potato puree should be eaten daily.

The detailed diet contains about no grams albumin, 105 grams fat and 200 grams
carbohydrates with a fuel value of 2247 calories.

The stool is best collected in quart fruit jars and examined as soon after evacua-
tion as possible. The wooden spatula like tongue depressors are well adapted to
handling the specimen.

THE

407

Having familiarized one's self with the degree of digestion of muscle, starch,
and fat in a normal person, we are in a position to judge of the state of assimilation
in a patient.

The first part of the test is the macroscopical one. For this grind up a faecal
mass of y% to i inch diameter in a mortar, gradually adding water until it has the
consistence of a broth. About % c.c. of this emulsion should now be squeezed
out between two slides and studied against a dark surface and then when held up
to the light. The normal stool gives a rather uniform brownish homogeneous layer.
Connective-tissue remnants (indicative of gastric derangement) show as whitish
fibers. Undigested muscle tissue remnants as reddish-brown splotches. Fat
particles as whitish-yellow clumps. Potato remnants appear like sago grains and
mash out easily like mucus. Mucus is best noted in the faecal mass before making
the emulsion. In the microscopical test of this emulsion:

We judge of muscle digestion by the intactness of the stria tions.
If a muscle remnant is only a homogeneous yellowish particle, it shows
satisfactory digestion. If it is rectangular, with well-defined cross
striations, it shows poor digestion for meat (Azotorrhoea). A loopful
of faeces should be smeared into a drop of Gram's solution for starch-
digestion determination. Normally there should be no blue-staining
starch granules.

Soaps are gnarled bodies everted like the pinna of an ear, while soap crystals
are comparatively coarse and do not melt on application of gentle heat as do the
more delicate fatty acid crystals. Neutral fat is in round or irregular globules. The
best stain for fat is Sudan in (saturated solution of Sudan in in equal parts of
70% alcohol and acetone).

Mix up the f ssces with dilute alcohol (50 to 70%) and then add a drop of the above
solution and apply a cover-glass quickly. The fat globules show as orange or
golden yellow bodies.

By rubbing up a small portion of the faeces in 36% acetic acid, applying a cover-
glass and heating over a flame until the preparation shows bubbles, we convert the
soaps and other fat combinations into free fatty acids which show as more or less
numerous highly refractile bodies showing a crystalline structure as the prepara-
tion cools. By practice one learns the amount of such globules to expect with differ-
ent fat contents in stools.

Steatorrhcea, or the presence of fat in abnormal quantities in the
faeces, is shown by the pale, bulky, greasy stools as well as in the micro-
scopical examination.

Average for normals in i gram dried faeces:

Total fat, 225 mg. (22.5%)

Total fatty acid, 86 mg. (37-9% of a11 fat)

Total soap, 74 • 7 mg. (33 . 4% °f aU fat)

Total neutral fat, 64 . 4 mg. (28 . 5 % of all fat)

408 BILE IN FAECES

In normal cases the only fat elements recognizable are yellow calcium or color-
less soaps. In sprue from 25 to 30% of the fat ingested appears in the stool while
the stool of pellagra shows only about 5% which is the normal figure.

As quantity of fat increases (as say 500 to 600 mg.) droplets of neutral fat appear
with or without increase in number of soap masses. Also needles and splinters
of fatty acid and soaps appear. Much connective-tissue debris shows defect in
gastric digestion, as only the stomach digests connective tissue.

A test for activity of fermentation should be made by using a Schmidt
apparatus.

A distinct evolution of gas in twelve hours shows starch digestion defect. Such
faeces are acid. A delayed production of gas (after twenty-four hours) shows albu-
min' decomposition. Such faeces show an alkaline reaction. The apparatus is
shown in Fig. 7. Into a stocky salt mouth bottle we put approximately 5 grams
of faeces which have been rubbed up into an emulsion with water and fill the bottle
with water. The remaining portion of the apparatus consists of a test-tube or a
graduated cylinder fitted with a doubly perforated rubber stopper. One U-shaped
glass tube passing through this stopper connects with a second test-tube. This tube
serves as a receptacle for any water which may come over from the water-filled tube
or graduated cylinder and has an opening punched out of the bottom of the test-
tube. The other opening in the twice perforated cork admits a straight tube which
connects with a large rubber stopper which fits into the bottle for the faeces. To
prepare, fill the graduated cylinder, then push in the doubly perforated cork which
is connected with the side receiving tube and the large rubber cork. This latter is
then pushed down to fit tightly into the bottle filled full with the faeces emulsion.

In addition to the faeces examination we should check the results from the test
diet with indican and nitrogen partition determinations of the twenty-four-hour
urine specimen — the ratio of ammonia nitrogen to total nitrogen indicating the func-
tional power of liver and the indican the question of stasis in lower part of small
intestine.

The most satisfactory test for bile in the faeces is to emulsify a small
particle of faeces in a saturated aqueous solution of bichloride of mercury,
preferably with a wooden toothpick, on a concave glass slide. After
one or more hours hydrobilirubin-containing faeces show a salmon pink
color and bilirubin ones a green color. One should familiarize himself
with these reactions in normal cases. The examination of faeces for
bile is less certain than duodenal fluid examination.

In examining a liquid stool after salts, it is well to color the drop of faeces, which
is to be covered with the cover-glass, with a small loopful of %% solution of neutral
red. If diluting fluid is used, it should be salt solution, and not water. The neutral
red tinges the granules of the endoplasm of amoebae and flagellates a very striking
rose pink color, thus differentiating them from vegetable cells or body cells.

Whether examining the thin faeces or the mucus particle, it is well to reserve
report on amoebae or flagellates until motion is observed. Encysted protozoa are

THE FAECES 409

difficult to diagnose. An experienced examiner easily recognizes the four or eight
nucleated cysts when the material is mounted in Gram's iodine solution.

When a smear preparation is desired, we may smear out a fragment of mucus
and stain by Romanowsky's or Gram's method. The character of the bacteria
present appears to be of diagnostic value— especially in the case of infants and young
children. Beautiful preparations may be made by mixing the faeces with water, then
centrifuging for one minute. This throws down vegetable debris and crystals. Now
decant the supernatant fluid, which holds the bacteria in suspension, and add an
equal amount of alcohol. Again centrifuge, decant, and smear out and examine
the bacterial sediment.

Simply taking a small mass of faeces and emulsifying it with a wooden
toothpick on a concave slide in 70% alcohol— then, after the sedi-
ment settles, taking up a loopful with platinum loop from the surface
and smearing out, gives a very satisfactory smear. Gram's method,
with dilute carbol fuchsin counterstaining, gives the best picture.

The Boas-Oppler bacillus may be found in the stools in this way. Normally,
a Gram-stained stool shows a great preponderance of Gram-negative bacilli and such
a finding in a measure excludes cancer of the stomach. Organisms which are Gram-
positive as well as the Boas-Oppler bacillus are, i. Lactic acid bacilli — these show
Gram- negative areas in the slender bacilli. 2. A type of bacillus similar in size to the
colon bacillus but Gram-positive and noncultivable (found in acid stools). 3. Bacilli
of the B. subtilis type.

It is very important to examine the faeces for T. B. With children a diagnosis
of tuberculosis may be made in this way when the sputum cannot be obtained,
the pulmonary secretion being swallowed. The preparation on the concave slide
as described above should be stained for T. B. Ulcerations in intestinal T. B. may
show very numerous bacilli.

To culture for typhoid, dysentery, cholera, or other bacteria, take up the material
in a tube of sterile bouillion and smear it out with a swab over a lactose litmus agar
plate or an Endo or Conradi-Drigalski plate. Before streaking the plates they
should be very dry on the surface. This can be best done by pouring into a plate
with a circular piece of filter-paper in the lid and placing in the incubator for one-half
hour to dry. The filter-paper absorbs the moisture. Then inoculate the surface of
the plate with the faecal material. Selective cholera plating media are strongly
alkaline.

In summer complaints of infants and children the organisms con-
cerned are as a rule related to various dysentery strains of bacilli.
Kendall in 293 stool examinations found the gas bacillus (B. Grog,
capsul.) in 22 cases. The gas bacillus produces intestinal disorders
which are not benefited by lactose but by buttermilk (lactic acid bac-
teria). For diagnosis, a loopful of the faeces is emulsified in a tube of
sterile milk or litmus milk. The emulsion is heated to 8o°C. and held
at this temperature for twenty minutes. After incubation for eighteen

410 GALL-STONES

to twenty-four hours, preferably anaerobically, we get (i) a shreddy
disruption of the casein, (2) the smell of rancid butter and (3) fully 80%
of the casein is dissolved. Smears show short thick Gram-positive rods
with slightly rounded ends. B. subtilis is sometimes found but does
not give a rancid odor nor the strong disruption of the clot.

It was until recently thought that Cammidge's reaction (urine) when associated
with azotorrhoea and steatorrhcea made for a diagnosis of chronic pancreatitis.
At present very little importance is attached to the Cammidge reaction.

Loss of weight, anaemia, diarrhoea and pains in the upper abdomen
are important indications of pancreatic trouble. As chronic pan-
creatitis is often associated with cholelithiasis jaundice is frequently
present. Glycosuria is not often present. While functional tests
are important they do not make for a sure diagnosis. At present we
examine the duodenal fluid for presence of pancreatic ferments.

Miiller's method for pancreatic functioning determination is to give a calomel
purge two hours after a meal. A little of the liquid stool is smeared on the surface
of blood-serum and the tube incubated at 6o°C. (paraffin oven). If the surface is
smooth, no trypsin was present; if dotted with spots of digestion liquefaction, it
shows that the pancreatic secretion is present.

In Schmidt's nucleus test small cubes of beef are hardened in absolute alcohol
and then tied up in tiny silk bags. These are recovered from the faeces and sec-
tioned. Complete preservation of nuclei indicates a total absence of pancreatic
functioning provided the passage of the tissue be not too rapid as by diarrhoea.

In the microscopic examination, epithelial cells are generally more or less disinte-
grated. In the mucus of bacillary dysenteric stools, however, large intact phago-
cytic cells are frequent, which may be mistaken for encysted amoebae.

Triple phosphate crystals are frequently observed in faeces, as may also be crys-
tals of various calcium salts. Charcot-Leyden crystals are rather indicative of
helminthiases.

Various flagellates, and in particular Lamblia, may be responsible for diarrhceal
conditions which may cause rather serious symptoms.

Balantidium coli has been reported several times as the cause of dysenteric con-
ditions. Coccidiadea are found in the faeces.

Isospora was not an infrequent finding in the stools of the soldiers at Gallipoli.

Gall-stones are usually recognized by their facetted appearance.
The stool should be examined for two weeks following an attack of hep-
atic colic and the faeces should be rubbed up in water and passed through
a seive. A concentric arrangement of layers is usually noted on frac-
turing a gall-stone. For identification dry and pulverize the stone
and treat with alcohol and ether. This dissolves the cholesterin and
upon evaporation the rhombic crystals separate out and may be recog-

THE FAECES

nized microscopically. The residue may be extracted with cold dilute
KOH solution. This extracts bilirubin which may be recognized by
Gmelin's test. Pseudo-gall-stones are usually masses of fats, soaps or
vegetable material.

At times enteroliths are mistaken for gall-stone. These are shells of inorganic
salts covering inspissated masses of faeces or seeds, etc.

It is in the faeces we examine either for the parasites or for their ova
in connection with practically all the flukes, except the lung fluke and
the bladder fluke; for intestinal taeniases and for practically all the
round worms, except the filarial ones.

Bass has recommended that faeces which have been made fluid be centrifuged
and the supernatant fluid containing vegetable debris poured off. The sediment
contains hookworm eggs. Then pour on sediment a calcium chloride solution of
sp. gr. 1050. Again centrifuge and decant. Next add calcium chloride solution
of a sp. gr. of 1250 and centrifuge. This brings to the surface the hookworm eggs
which may be pipetted off. As a rule, the finding of hookworm eggs is very easy
without such a technic. The eggs of Trichostrongylus greatly resemble those of
hookworm but are larger, 73 to gi/i long. In perfectly fresh faeces Strongyloides are
present as worm-like embryos while hookworm gives only two to four segment eggs.

In the tropics, the examination of the faeces vastly exceeds in value
that of urine and is possibly more important than blood examinations.

The larvae of various insects may at times be detected in the stools, as well as
certain acarines (cheese mites, etc.).

The test for occult blood is indicated in helminthiases as well as in the conditions
for which it is usually tested.

CHAPTER XXIX
BLOOD CULTURES AND BLOOD PARASITES

CLINICALLY, the most important examinations of the blood for
parasites is for the presence of various bacterial infections and for
certain blood protozoa and also filarial embryos.

The modern method of culturing blood, especially for the detection of typhoid
or paratyphoid bacilli, is by the use of the bile media of Conradi. Test-tubes are
filled with 7 to 10 c.c. of i% peptone ox bile, or ox bile alone, and the medium is
sterilized in the autoclave. It is good practice to place the syringe in a plugged
test-tube containing salt solution, with the needle unscrewed. After autoclaving,
the sterile syringe can be taken to the bedside in the test-tube. Using a wide test-
tube, a forceps can be sterilized at the same time and used to attach the needle
to the barrel of the syringe.

By using a piece of glass tubing into which the needle is inserted we may sterilize
the syringe easily in the test-tube. The glass tubing prevents the steel needle from
coming in contact with the glass of the test-tube and so prevents cracking the test-
tube.

The skin should be scrubbed gently with green-soap solution and water for about
three minutes. The skin of the area to be punctured should then be sterilized by
the gentle application of Harrington's solution (not scrubbed) for one-half minute,
and should then be washed with sterile water. It appears to be safe to simply
scrub the area with 70% alcohol for one or two minutes. Applications of pure
carbolic acid on a gauze wad for a few seconds followed by neutralization with 70%
alcohol gives satisfactory sterilization. The present method of sterilizing skin
for taking blood or inoculating vaccines is simply to smear the site of entrance for
the needle rather heavily with tincture of iodine. .A tourniquet is now applied to
distend the vein, and the needle, beveled side up, is inserted in the direction of the
venous flow. Withdrawing 5 to 10 c.c. of blood, we loosen the tourniquet (otherwise
the blood may flow from the puncture) then withdraw the needle, and force out
about ^ c.c. into the first bile tube, about i c.c. into the second, and 2 or 3 c.c.
into the third. It is well to reserve some of the blood for Widal tests.

The bile tubes are now incubated for ten to twelve hours and then transfers
are made to bouillon tubes. These bouillon tubes can be used in six to eight hours
for testing the organism against knawn typhoid or paratyphoid sera. Test-tubes
containing 10 c.c. of ordinary bouillon with i% of sodium citrate are as satisfactory
as bile media.

Some prefer a 2% sodium glycocholate in bouillon while others use a 2% solution
of ammonium oxalate in bouillon for blood cultures.

Some prefer to streak plates of lactose litmus agar with material from the bile

412

BLOOD CULTURES AND BLOOD PARASITES 413

tubes instead of inoculating the bouillon tubes. Contamination with staphylococci
or the presence of staphylococci, streptococci, or plague bacilli in septicsemic con-
ditions show easily accessible colonies.

Schotmuller adds i to 3 c.c. of blood to liquefied agar at 45°C., and after mixing
pours into plates. The standard method formerly was to add the blood to an
excess of bouillon (i to 5 c.c. of blood to 100 c.c. or more of bouillon).

The method of culturing blood we now follow in our laboratory is
the following:

A stout hypodermic needle is attached to about 6 inches of rubber tubing which in
turn is pushed over a downward bent glass tube which passes through a doubly
perforated rubber stopper. A second glass tube, which also passes through the stop-
per, is bent upward to be attached to a second piece of rubber tubing for use in suc-
tion by the mouth. The glass tubes project about Y^ inch below the undersurface
of the rubber stopper and above are about 2% inches including the bent arm. This
system of tubing and stopper is readily sterilized by boiling in a pan or instrument
sterilizer. As a receptacle for the blood we employ Erlenmeyer flasks of 100 c.c.
capacity, containing 10 to 25 c.c. of salt solution with i or 2% of sodium citrate, for
prevention of coagulation. These citrated salt solution flasks are plugged with cot-
ton, sterilized and kept on hand ready for immediate use, so that we only have to
sterilize the stopper and tubing by boiling and flame the neck of the flask when re-
moving the cotton plug to insert the stopper of the system. By suction we can take
any amount of blood desired. I usually count the drops of blood as they fall into
the citrated salt solution allowing 16 drops to the cubic centimeter. In this way we
may take from 10 to 25 c.c. of blood at the bedside and then later on in the laboratory,
when it is convenient, inoculate various media from the flask. For plates add 2 or 3
c.c. of this citrated blood to 6 or 8 c.c. of melted agar at 45°C. The blood mixture
can also be added to various sugar bouillons for fermentation reaction. Finally we
place the receiving flask in the incubator and culture it as well as the other media.

Of course in inoculating the various plating or sugar tubes from the flask there is
some liability to contamination. This may be avoided by removing with a sterile
pipette 10 to 20 c.c. from the flask containing the citrated blood to carry out the
inoculations instead of pouring out directly from the flask.

A very useful procedure in the isolation of streptococci, pneumo-
cocci, plague and anthrax bacilli is to inject i to 2 c.c. of blood into
suitable animals. When infecting mice use only about 0.2 c.c. sub-
cutaneously at root of tail or, more certain of results, the injection of
about i c.c. of the blood intraperitoneally. Streptococci, even from
virulent human infections, are uncertain in their action on animals
so that the failure to produce septicaemia in the mouse does not neces-
sarily indicate that the organism is of slight virulence.

By using the bile media, we can take the blood from the ear in typhoid cases,
if preferred. Then if chance staphylococcic contamination occurs, such colonies
are readily differentiated from typhoid ones by the pink color on lactose litmus

414 BACTERIAEMIA

ITS be

agar. For culturing blood in septicaemic conditions, the blood should always
drawn from the vein and cultured either by mixing i to 2 c.c. with melted agar
and then pouring plates or by transferring to bouillon in excess (at least ten times
as much bouillon as blood) and after eighteen to twenty-four hours' incubation
plating out. For Streptococcus and Pneumococcus blood agar plates are to be pre-
ferred, the Pneumococcus giving green colonies with only a suggestion of haemolysis
while the Streptococcus gives an opaque colony with a distinct haemolytic zone
surrounding it. We rarely culture blood anaerobically as the important patho-
genic anaerobes (tetanus, gas gangrene and malignant oedema) do not tend to
invade the blood stream during life. Recently a case has been reported where the
gas bacillus was isolated from the blood of a soldier with gas gangrene. There is
an anaerobic streptococcus S. putridus which grows anaerobically on blood agar giving
porcelain white colonies without haemolysin. The cultures have a putrid odor.
Not pathogenic for animals.

Warren and Herrick have recently published a very important study
of 134 cases of bacteriaemia. Bacteriaemia signifies the mere presence
of bacteria in the blood without reference to symptoms, while sepsis
denotes conditions due to invasion of the blood stream by bacteria or
their toxins with marked systemic reaction.

Of 25 cases with endocarditis 22 died and 3 were unimproved. Of 55 cases of
sepsis 39 died and 10 recovered. In postpartum infections 10 died and i recovered.
In osteomyelitis 5 died and 4 recovered. In otitis media 2 died and 4 recovered.

Thirty-one cases were due to Streptococcus hamolylicus of which 21 died.

Forty cases were due to S. viridans and 25 died. One case from S. mucosus died.

Of 39 cases with Staphylococcus aureus 22 died and in 3 cases of S. albus
2 died.

Of 10 cases of Pneumococcus bacteriemia 6 died. Six of B. coli infection gave 4
deaths; two of B. influenzas 2 deaths; three of anaerobic streptococci, 2 deaths and
two of B. mallei, 2 deaths.

Seven cases of mixed infections gave 6 deaths.

The ordinary leukocyte and differential count procedures were of very little value
in prognosis.

The average white count in fatal cases of S. hamolyticus infection was 18347
with 81% of polymorphonuclears while in cases recovering it was 18742 and 85%,
respectively. Fatal S. viridans infections gave an average white count of 15976 with
polymorphonuclear percentage of 78, while nonfatal cases gave 17222 and 75%,
respectively.

Of 25 cases treated with vaccines, 81% died; while of 47 under surgical treatment,
50% died and with 50 treated pallia tively, 50% died.

Typhoid cultures are best obtained in the first week of the disease,
after that time the Widal is the test of preference.

If a paratyphoid serum is not at hand for testing, it may suffice to inoculate a
glucose bouillon tube or a Russell lactose glucose litmus slant; gas production
indicates paratyphoid. This test should be applied when a very motile organism

BLOOD CULTURES AND BLOOD PARASITES 415

does not show agglutination with a known typhoid serum. Anthrax and glanders
should be considered in blood cultures.

In Malta fever it must be remembered that colonies do not show themselves
for several days. Addition of blood to melted agar is a good procedure.

Blood for culturing typhoid or the paratyphoids may be taken with
a Wright's tube from the ear or finger. Dipping the hand in hot
water assists the flow of blood. The supernatant serum after centrif u-
galization should be pipetted off with a sterile pipette and reserved
for agglutination tests while the clot is dropped into a bile tube. (Clot
culture.)

Rosenberger was the first to insist upon the importance of examination of blood
for T. B. Brem considered that many cases of finding of acid-fast bacilli were not
of T. B. The Kurashigi-Schnitter method for tubercle bacilli in blood is to take
about i c.c. blood and put in a centrifuge tube containing 5 c.c. of 3% acetic acid.
After the red cells are thoroughly laked centrifuge, pipette off supernatant fluid and
dissolve the sediment in 5 c.c. antiformin. When dissolved add 5 c.c. absolute
alcohol and centrifugalize for twenty minutes. Smear out the sediment and stain.

The examination of the blood for the parasites of malaria, filariases, kala-azar
and spirillum fevers has been discussed under their respective headings.

With trypanosomes from human trypanosomiasis, smears from gland juice or
cerebrospinal fluid seem more satisfactory to examine then blood smears unless the
blood is taken in 5 to 10 c.c. quantities and centrifuged in sodium citrate salt
solution.

The latest method in the diagnosis of trichinosis is to take 5 to 10 c.c. of blood
from a vein at the time of the migration of the embryos to the muscles (ten to twenty
days). This is forced out into a centrifuge tube containing 3% acetic acid, and
the sediment examined for trichina larvae.

CHAPTER XXX
THE STOMACH AND DUODENAL CONTENTS

FROM a microscopical standpoint there is comparatively little that
is of value in the examination of the gastric contents; there is nothing
very specific about the findings.

A test meal is not a necessity as in the chemical examination, but
either vomitus or material withdrawn with a stomach-tube two or more
hours after an ordinary meal suffice.

The most satisfactory specimen is one taken before the giving of
the test meal.

The washings from the stomach are allowed to stand until the
sediment has fallen to the bottom and an examination of this is made.

The microscopical diagnostic points in connection with distinguishing cancer of
the stomach from nonmalignant dilatation are : i . Fragments of cancer tissue. These
are very rarely found and are most difficult to diagnose. 2. The presence of flagell-
ates in the early stages of cancer (the so-called anacid stage preceding the develoy-
ment of lactic acid). As flagellates prefer an alkaline medium, they disappear
after the acidity due to lactic acid comes on. 3. The presence of the Boas-Oppler
bacillus. There are probably several organisms so designated. They are lactic acid
producers and are characterized by being very large bacilli (7XI/-0 and arranged in
long chains which stretch across the field of the microscope. They are Gram-posi-
tive and do not form spores. They can be cultivated on media rich in milk or blood
and are aerobic. They should only be reported when present in great abundance and
in long chains. Heinemann thinks it probable that the Boas-Oppler bacillus, Lepto-
thrix buccalis, and B. bifidus may be identical with B. bulgaricus (see under Milk).

4. The absence of sarcinae and yeasts. The presence of these sarcinae and fungi in
vomitus is indicative of a simple dilatation.

In the diagnosis of cancer, other than the finding of tumor, gastric
stasis, etc., much importance is attached to the absence of free HC1.
This is best determined by the Gluzinski test. In the Mayo clinic
total acidity of gastric contents averaged 63 (% free HC1) in ulcer,
while cancer averaged only 31 (% free HC1). Lactic acid was found
in 43% and blood, occult or otherwise, in 73% of cancer cases.

In determining the pepsin activity of the gastric juice we usually
employ the Mett tube.

416

THE STOMACH AND DUODENAL CONTENTS 417

In chronic gastritis the picture of mucus entangling large numbers of cells is
characteristic. It must be remembered, however, that strands of mucus entangling
polymorphonuclear cells may be found in normal gastric contents. The pus cells
which are free are the ones of significance. An occasional cylindrical gastric epithe-
lial cell may be found. The recognition of carcinoma cells, especially those showing
mitoses, is of value only when done by one who has made a special study of the
findings.

In examining the sediment from the filter-paper after filtering off the stomach
contents always use a dilute Gram solution (about i to 4) for mounting the sediment.
Muscle fibers, yeast cells, red blood-cells, and epithelial cells are stained a golden
yellow. Starch granules are stained blue while fats are unstained and show as
globules of varying sizes.

The Duodenal Fluid.— This is best obtained with the Jutte modi-
fication of the Einhorn duodenal tube. There is little difficulty
in the passage of the tube when the stomach is not dilated. One can
aspirate from 5 to 20 c.c. in about five minutes, the amount varying with
the individual case. The normal fluid is clear, bile stained, sero-
mucous and contains few cells and living bacteria. Dead bacteria may
be found in considerable numbers.

Of the greatest importance is the chemical examination of duodenal fluid for
pancreatic ferments. It is well recognized that the examination of the faeces for
such ferments is absolutely unreliable while with duodenal fluid it is most satisfac-
tory. The normal ferment value of duodenal fluid is best indicated by the tryptic
power, this being very constant. The lipase findings are very variable.

Normally 10 c.c. of 0.1% casein solution is digested in twenty-four hours by
duodenal fluid in dilutions of i to 3000 to i to 10,000 or even higher.

Turbidity of the fluid is thought to be suggestive of cholelithiasis.
Bile-stained pus cells are also of significance in cholecystitis. Hence
examine sediment unstained before adding Gram's iodine solution.
The presence of bile in the duodenal fluid is a far more certain index
of the patency of the bile duct than the test of faeces for bile as bile
may be absent in a fasces test when present in duodenal fluid. The
color of the fluid is about as satisfactory an indication of the presence
of bile as that given by the various tests for bile. Einhorn states that
with turbid bile cholecystitis with gall-stones is almost always present
if with fasting condition. Gall-stones without cholecystitis may give
a clear fluid Liver conditions, as neoplasms or high grade cirrhosis
may give rise to turbid duodenal fluid.

McNeil attaches importance to the Wolff- Junghan's test of the duodenal fluid
which he states gives turbidity up to the third or fourth tube, but never in the fifth
or sixth one, normally. An increase would indicate some pancreatic, biliary or
duodenal inflammation, provided we checked up the test on the gastric contents.
27

4i8

DUODENAL FLUID

In duodenitis we have stringy mucus and rather considerable numbers of livii
bacteria.

Typhoid carriers are best recognized by culturing the duodenal contents. Mac-
Neal, in culturing the organisms from the duodenal contents of 26 cases, found that
few living bacteria were present although dead ones might be present in considerable
numbers. The living organisms are as a rule Gram-positive cocci.

B. lactis aero genes was found rather constantly by Gessner.

In connection with the possible significance of bacteria in the duo-
denal contents it is interesting to note the findings of Kelly in a bac-
teriological study of 413 cases of operations on the biliary tract by
Deaver. About one-half the cases showed sterile bile but in the acute
cases living bacteria were almost constantly present. B. coli was
found in 30%, B. typhosus in 7%, Staphylo coccus pyogenes in 2.9%,
Streptococcus in 0.2%. Other organisms were more rarely found and
55% were sterile.

In recently studying the bacteria of the duodenal contents and subsequently t
bile of a case of cholelithiasis with cholecystitis the only organism found was B. coli.
This organism was markedly haemolytic on blood agar plates.

For the carrying out of the various tests see appendix.

CHAPTER XXXI
EXAMINATION OF PUS

Pus may be collected for examination either i. with a platinum loop,
2. with a sterile swab, 3. with a bacteriological pipette or 4. with a
hypodermic syringe.

It is always well to make a smear and stain it by Gram's method at
the same time that cultures are made. The Gram stain gives informa-
tion as to the abundance of organisms in the pus and as to the probable
findings in the culture. Pneumococci and streptococci are differenti-
ated from the staphylococci in this way without the necessity of more or
less extended cultural methods.

Smears from material examined for gonococci may show Gram-negative diplo-
cocci which, however, do not generally have the morphology of the Gonococcus.
They are furthermore extracellular.

The M. catarrhalis has been reported from urethral smears though very rarely.
Diphtheroid organisms are not uncommon. Gram-positive cocci are rather common
in smears from discharges of chronic gonorrhoea.

When autogenous vaccines are to be made, the isolation of the exciting organism
is necessary. This is best done by streaking the pus, taken up with a sterile swab
and emulsified in a tube of bouillon, over the surface of an agar plate. Practically
as convenient and providing a more nutritious medium is to smear the material on
a loop or swab over the surface of a blood-serum slant, then to inoculate a second
tube from the first without recharging the loop or swab, and so on until three or four
tubes are inoculated. Isolated colonies should be obtained in a third or fourth tube.

In examining blood-serum slants inoculated with purulent material, always
examine the water of condensation for streptococci.

A bacteriological pipette is very useful when pus is to be sent to a laboratory;
the tip can be sealed in a flame and the cotton plug at the other end insures the
noncontamination of the contents. The material may be drawn up either with the
mouth or with a rubber bulb.

The hypodermic syringe is very useful in puncturing buboes, etc.,
especially in plague. A small pledget of cotton on a toothpick dipped
into pure carbolic acid and touched to a spot over the bubo, which after
about thirty seconds is soaked with alcohol, makes a sterile anaesthetic
spot at which to introduce the needle of the syringe. It must be remem-
bered that when plague buboes begin to soften, the plague bacilli may

419

420

BUCCAL AMCEB.E

be replaced by ordinary pus organisms. The pus from wounds in-
fected with anaerobes is usually very foul. The most important anae-
robe in the discharge from gas gangrene wounds is B. perfringens.
The pus from the necrotic center of climatic bubo is sterile.

It is remarkable how frequently we get pure cultures from abscess material. In
purulent material from abdominal abscesses we are apt to obtain mixed cultures,
especially the colon bacillus and B. pyocyaneus, in addition to ordinary pus organisms.

When it is a question between streptococci and pneumococci, it is well to inocu-
late a mouse; the capsulated pneumococci at the autopsy make the diagnosis.

Animal inoculation is also necessary in plague and glanders, and possibly anthrax.
When tetanus is suspected, it should be examined for as described under Tetanus.
Tuberculosis should also be identified by inoculating a guinea-pig, as well as by acid-
fast staining and culture, if there is any doubt as to the nature of the material.

The black or yellow granules of madura foot, as well as those of actinomycosis,
should be examined as recommended in the section on fungi.

Amoebae, coccidia, and larval echinococci may be found in purulent material,
as may also various other animal parasites, as fly larvae, sarcopsyllae, etc.

The pus from an amoebic abscess of the liver is as a rule sterile when cultured.

The examination at the time of operation or exploration frequently
shows an absence of amoebae as well as of bacteria. Two or three days
later amoebae may be found in the pus draining from the abscess cavity.

Flukes, round worms, and whip worms may as a result of their wandering from
the intestinal lumen cause abscesses.

Serious ulcerations may follow infection with the Guinea-worm.
Abscesses of filarial origin are to be thought of.

CHAPTER XXXII
SKIN INFECTIONS

CULTURAL methods are as a rule to be preferred in the bacterio-
logical examination of the skin.

This is best done by washing the surface to be examined with soap and water,
in order to eliminate chance organisms which may have settled on the surface of
the skin in dust or as a result of contact with material containing them. Scrapings
are then made with a sterile dull scalpel, and this material is emulsified in a drop of
sterile water in the center of a Petri dish. A tube of melted agar at 42°C. is then
poured on the inoculated drop and, by mixing, the bacterial flora is distributed over
the entire surface of the plate. Of the colonies developing on such plates probably
80% will be found to be staphylococci, and of these the greater proportion will
be staphylococci showing white colonies.

Occasionally the aureus or citreus may be isolated.

Streptococci and colon bacilli are rarely found.

The Staphylococcus pyogenes aureus is the organism usually isolated from furuncles,
circumscribed abscesses, and carbuncles.

Streptococci are the organisms to be expected in phlegmonous infections.

Cold abscesses, which are frequently due to tuberculous infection, are, as a rule,
sterile.

Acne pustules may show staphylococci or the microbacillus of acne may be present.

The Bacillus acnes is a short broad bacillus often showing a beaded
appearance when stained by Gram's method. It is Gram-positive.
According to Hartwell it grows readily on glucose agar when cultivated
anaerobically (Wright's method). Colonies appear in four to five days.

Sabouraud's medium for its culture is: Peptone 20 grams, glycerine 20 grams,
glacial acetic acid 5 drops, agar 15 grams and water 1000 c.c. The bottle bacillus,
which morphologically resembles a yeast, is considered to be the cause of dry pityri-
asis capitis. It may also be found in the comedones of children.

In the tropics, an organism which at times produces lesions similiar to impetigo
and again pemphigoid eruptions and at other times widespreading erysipelatous
conditions gives cultural characteristics similar to S. pyogenes aureus. It is probably
only a virulent aureus. It has been described under the name of Diplococcus pem-
phigi contagiosi.

The Staphylococcus epidermidis albus, or stitch abscess coccus, is considered by
Sabouraud to be the cause of eczema seborrhoicum.

It is in scrapings from the skin of lepromata that we find acid-fast
organisms in the greatest profusion. In tuberculosis of the skin the

42 T

422

THE SKIN

tubercle bacilli are exceedingly scarce. Inoculation of a guinea-pig
will probably give positive results with the tubercle bacillus. The
leprosy bacillus is noninoculable for experimental animals.

Anthrax and glanders cause skin lesions which can only be surely diagnosed
culturally or by animal inoculation.

Plague bacilli may be isolated from the primary vesicles appearing at the site of
the flea bite.

Tropical phagedaena is thought by some to be due to a sort of diphtheroid or-
ganism. The organisms of Vincent's angina may cause tropical ulcer. Herpes
zoster has been reported by Rosenow as most probably due to a streptococcus with
special affinity for the ganglia and posterior roots.

The skin diseases due to fungi are discussed under that section. Of the skin
affections caused by animal parasites, ground itch is the most important. This is a
form of dermatitis due to the irritation set up by the hook-worm larvae penetrating
the skin of the foot and leg.

The Sarcopsylla penetrans or jigger (sand flea) is an important agent in ulcera-
tions about the foot.

Certain acarines cause skin lesions, as is also the case with the larvae of certain
flies.

'fhe itch mite (Sarcoptes scabiei) is an important animal parasite of the skin.

The various lice, fleas and bedbugs are well understood as causes of skin irrita-
tion.

Filarial infections are also important especially the ulcers of the
Guinea-worm, Calabar swellings of F. loa, the cystic tumors of F.
volvulus and the varicose groin glands and elephantiasis of F. bancrofti.

Leeches, as H. ceylonica, may cause serious ulceration.

Oxyuris may cause a severe irritation about the region of the groin and inner
surfaces of the thigh, and especially about the vulvar region of female children.

Gnathostomum siamense, a nematode with two lip-like structures and spine-
like appendages covering its anterior one-third, has been found once in a tumefac-
tion of the breast.

Plerocercoid larvae of Dibothriocephalidae have been found in the subcutaneous
tissues.

Certain skin diseases, as Oriental sore and yaws, are protozoal in origin. The
cutaneous lesions of uta or espundia are now known to be caused by a Leishmania
as well as Oriental sore. These affections in the Central and South American
countries are now known as American leishmaniases.

Dew itch or foot itch is caused by the penetration into the skin about the toes of
the strongyloid encysted larva of the hook-worm.

CHAPTER XXXIII
CYTODIAGNOSIS AND SPINAL FLUID EXAMINATIONS

THIS method of diagnosis is chiefly employed in the examination of
cellular sediments of pleural, ascitic, and cerebrospinal fluid.

The fluids which pathologically collect in the serous cavities are
divided into two classes, i. the transudates, which form as the result
of some circulatory inadequacy and 2. the exudates, which result from
inflammatory processes.

Transudates have little or no fibrin and very few cellular elements and do not
contain nucleo-albumin. Exudates contain nucleo-albumin and usually have a
specific gravity above 1018, while that of the transudates is lower than 1018.

There are two simple methods for differentiating transudates and exudates.
Moritz adds 2 drops of a 5% solution of acetic acid to the fluid to be tested. A
heavy, cloud-like precipitate shows the fluid to be of inflammatory origin (an
exudate). A transudate may produce a slight opalescence. Rivalta's test consists
in dropping a drop of the fluid to be tested into a cylinder containing 2 drops of
glacial acetic acid in 100 c.c. distilled water. A nebulous cloud as the drop of fluid
sinks shows an exudate.

For pleural fluids we should receive the material in centrifuge tubes about one-
fourth filled with 2% sodium citrate salt solution. This prevents clotting. Having
thrown down the sediment, the supernatant fluid is poured off, and in its place a i%
aqueous solution of formalin is added. After mixing and allowing to stand for about
five minutes, centrifugalization is again repeated and, pouring off the supernatant
formalin solution, we make smears from the sediment. This is either stained by a
Romano wsky method or, after fixing with heat (burning alcohol), the smear is
stained with haematoxylin and eosin.

With ascitic fluid it is usually sufficient to centrifuge the fluid, then decant off the
supernatant fluid and drain by means of a piece of filter-paper held at the mouth
of the upturned tube. The sediment adheres to the bottom of the tube and is
best emulsified with the small amount of fluid remaining by means of a bulb pipette.
The material is sucked up, smeared out on a slide with a second slide as for blood
and stained preferably with Giemsa after fixation. H. E. staining brings out
mitotic figures best. If the fluid has coagulated it is best to take a little of the
coagulum and stain it with neutral red as for vital staining. It is difficult to disso-
ciate the cells from the clot. I now make it a rule to collect a portion of the pleural
or ascitic fluid in citrated salt solution in order to prevent coagulation. The mate-
rial is then centrifuged and after removal of supernatant fluid with a bulb pipette
the cell sediment is drawn up and smeared out on a slide for cell or bacterial staining.

423

424 CYTODIAGNOSIS

By making smears as for blood beautiful preparations may be obtained. I prefer
Giemsa for differentiating cells and Gram's staining for bacteria.

The wet Giemsa method described for blood gives good results with puncture
fluid sediments.

At the time of securing fluid for cytodiagnosis, cultures should be made on blood-
serum for various pyogenic bacteria and, if tuberculosis is suspected, inoculation
of a guinea-pig is indicated.

The interpretation of cellular sediments is more difficult than many
books would indicate, there being many factors which tend to compli-
cate the findings.

The polymorphonuclears in purulent fluids often show fatty degeneration, swollen
and faintly staining nucleus or a breaking up of the nucleus into small deeply
staining masses (nuclear fragmentation). Such fragments in the smear may be con-
fusing. The endothelial cells often show fatty degeneration in the cytoplasm and
we often note bacteria and other cells which have been phagocytized by them.
Where proliferation of endothelial cells is going on actively the cells show a rather
deeply staining cytoplasm as compared with the light staining cytoplasm of the cells
in transudates. Some authorities attach importance to the Foulis' cells in connec-
tion with malignant processes in the peritoneum; often those associated with malig-
nant types of ovarian cysts. Such cells are large, often multinucleated and may
show appearance as if budding.

The following are the leading differentiations:

1. A smear showing almost entirely lymphocytes with a few red
cells and very rarely a polymorphonuclear indicates a tuberculous
process.

2. Where a pyogenic process is engrafted on a tuberculous one, we
have still the red cells, some degenerated lymphocytes, and in particular
polymorphonuclears showing fragmentation of their nuclei.

3. When a hydrothorax results from chronic hear tor kidney disease,
the characteristic cell is the endothelial cell, which greatly resembles a
large mononuclear. These cells often are arranged in plaques.

4. Some authorities consider that the cancer cell can be recognized
by its occurring in masses and having a markedly vacuolated cytoplasm.
It has been claimed that they contain glycogen by which means we can
distinguish them from endothelial cells which they so much resemble.
If such cells should show mitosis the finding would be suggestive.
For mitotic figures wet fixation with some bichloride fixative, with H. E.
staining, is best.

Jousset introduced inoscopy as a means of diagnosing tuberculosis. The fluid
was allowed to coagulate and was then digested with an artificial gastric juice. The
digested material was then centrifuged and the sediment examined for tubercle

CYTODIAGNOSIS AND SPINAL FLUID EXAMINATIONS 425

bacilli. This process does not seem to have met with much favor in this country.
(Using sodium citrate obviates the necessity for digesting the coagulum.)
The same points will hold for ascitic fluid as for pleural fluid.

CEREBROSPINAL FLUID EXAMINATIONS

In taking cerebrospinal fluid for culture and cytodiagnosis we use a
stout antitoxin needle without attaching a syringe. Aspiration is
responsible for many of the ill effects of lumbar puncture.

The needle should be about 4 inches long for an adult. Sterilize the skin and needle
as described for blood cultures from a vein. To make a lumbar puncture, place
patient on left side with knees drawn up. A line at the level of the iliac crests passes
between the third and fourth lumbar vertebrae. Select a point midway between the
spinous processes of these lumbar vertebrae and enter the needle two-fifths of an
inch to the right of this point, pushing the needle inward and upward. Collect
the material in a sterile test-tube. Make cultures on blood-serum and then cen-
trifugalize and examine the sediment as for pleural fluids.

Cell Count. — A method of examination considered by neurologists
as of differential diagnostic value is to count the number of cells in a
cubic millimeter of the cerebrospinal fluid. The technic is to use a
gentian- violet- tinged 3% solution of acetic acid. This is drawn up to
the mark 0.5, and the cerebrospinal fluid is then sucked up to n.
After mixing, the cell count is made with the haemocytometer. Nor-
mally we have only one or two cells per cubic millimenter, but in tabes
or general paresis this is increased to 50 or 100 cells (greatest at onset of
disease).

It is advisable to make the cell count of the fluid as soon after obtaining it as
possible, the cells tending to degenerate. It is customary to consider fluid contain-
ing blood as unsatisfactory for the cell count as well as for the globulin tests, but one
can calculate the leukocytes due to blood content by counting the red cells and sub-
tracting one leukocyte for each 750 red cells.

It is now generally recommended to make the spinal puncture with the patient
seated on a stool with the shoulders inclined forward thus giving the greatest space
between the spinous processes. After the punpture the patient should drink a
glass or so of water and remain in bed for a day. In some clinics the subjects lie
down for a few hours and then return to their homes.

In general terms it may be stated that:

1. A lymphocytosis indicates a tuberculous or poliomyelitis process.

2. An abundance of polymorphonuclear and eosinophilic leukocytes
indicates a meningococcic, streptococcic, influenza or pneumococcic
infection. The fluid in (i) tends to be clear, that in (2) cloudy.

426

LANGE'S PARESIS TEST

When the case is one of meningism there are very few cells. In poliomyelitis
there is a cell increase of which 90% may be lymphocytes.

Trypanosomiasis gives a cellular increase very similar to syphilis.

In the work of the French Sleeping Sickness Commission five cells per cubic
millimeter was taken as normal.

Miller gives the following table as to pleocytosis:

AVERAGE INCIDENCE OF LYMPHOCYTOSIS IN THE SPINAL FLUID
(Plaut, Rehm and Schottmuller)

CLINICAL DIAGNOSIS
Cerebrospinal lues

FREQUENCY
85-90%

REMARKS

Counts often over 100 — may
c.mm.

-

reach 1000 per

Tabes dorsalis

00%

Counts usually under 100.

General paresis

08%

Counts average 30—60 cells pe

r c.mm.

Secondary lues

7Q— AO%

Moderate increase as a rule.

Multiple sclerosis

25%

Border-line counts.

Cerebral haemorrhage .
Cerebral tumors
Sinus thrombosis

f Frequency
is
[ variable

Cellular increase is apt to be a
one.

very moderate

Colloidal Gold Test (Lange's). — It is now generally accepted that
this test is more diagnostic of general paresis than any other single
test. The color changes in the first five tubes (i-io; 1-160) are so
constant that the term "paretic curve" is applied to such findings.
Of less diagnostic value are the so-called cerebrospinal lues curves
where the color changes, though of less intensity than the paretic ones,
are most marked in the third, fourth, fifth and sixth tubes (1-40 to 1-320).
In various types of meningitis, other than luetic, the color changes are
at times more marked in the tubes with the higher dilutions of spinal
fluids (from 1-320 to 1-2560).

The paretic curve of the colloidal gold test generally runs parallel with a spinal
fluid Wassermann and globulin increase. This agreement does not exist at all con-
stantly for positive blood-serum Wassermann tests and increased cell counts.

It may be stated that this test is of more importance in paresis than any single one
of the four reactions of Nonne, viz. (a) blood-serum Wassermann (b) spinal fluid
Wassermann (c) globulin increase and (d) increased cell count of spinal fluid (pleo-
cytosis). Of course, all of these tests should be carried out.

CYTODIAGNOSIS AND SPINAL FLUID EXAMINATIONS 427

Test. Put 1 1 clean dry test-tubes in a rack and deposit in the first tube 1.8 c.c.
of a 0.4% solution of sterile saline. Into the other 10 tubes put only i c.c. of the
0.4% saline. Into the first tube deliver 0.2 c.c. of spinal fluid and mixing
thoroughly we have 2 c.c. of a i-io dilution. Withdraw i c.c. from the first tube
and add to the i c.c. of saline in the second tube. This gives 2 c.c. of 1-20.
Continue the process until the No. i to No. 10 tubes contain i c.c. quantities of the
various dilutions from i-io to 1-5120.

Tube ii contains no spinal fluid but only i c.c. of the saline and serves as a
control.

To each of these n tubes add 5 c.c. of the colloidal gold reagent. The color
changes are usually read after the tubes have stood over night at room tem-
perature. The proper color of the control in tube n should be salmon red or old
rose and the fluid should be perfectly transparent. When the color is changed in
tubes containing dilutions of the spinal fluid we record one showing a bluish tint
as i. When the change is to a lilac we record it as 2. A distinct blue is marked
as 3 and a pale blue as 4. When decolorization is complete there is the highest
color change, which is noted as 5.

All glass-ware used in the test should be thoroughly washed in soap and hot water
and then carefully rinsed with tap water. Next use the bichromate-sulphuric acid
cleansing fluid, followed by most thorough washing in running water followed by
distilled water.

In preparing the reagent a 2 liter glass beaker, following the above-described
cleansing, is rinsed in double or triple distilled water, made with block tin condens-
ing tubes and without rubber connections. Then fill the beaker with triple dis-
tilled water up to a ^ liter mark. Heat the water gradually to 6o°C. Now
add 5 c.c. of a i% aqueous solution of Merck's yellow crystalline gold chloride
and 3^ c.c. of a 2% aqueous solution of desiccated potassium carbonate. Con-
tinue the heating of the solution, which should remain clear, to 8o°C., then
add 5 drops of a i% aqueous solution of oxalic acid, stirring all the time. The solu-
tion should be colorless after adding the oxalic acid. When the temperature reaches
QO°C. remove the flame and add drop by drop 5 c.c. of i% formalin solution,
stirring continuously. Should a pink color show itself before all the formalin solu-
tion has been added stop the further addition. It soon assumes the required shade
and when cool should be perfectly transparent and of an old rose or orange-red color.

Globulin Increase Tests. — The test generally used is Noguchi's buty-
ric acid one. Deliver into a small test-tube 0.5 c.c. of a 10% solution
of butyric acid in 0.9% salt solution. Then add o.i c.c. of spinal fluid.
Bring to a boil over a flame and add o.i c.c. of N/i NaOH solution.
If there is a considerable increase of globulin a flocculent precipitate
appears in a few minutes or at any rate in one or two hours. Fluids
with a normal content or only slight increase only show a slight opacity.

The odor of the butyric acid is very objectionable and in our laboratory we use
the Ross-Jones method. In this one deposits in a small tube about i c.c. of satu-
rated solution of ammonium sulphate. On the surface of this column we deposit
i c.c. of spinal fluid. If globulin increase is present a turbid ring appears within

428

GLOBULIN INCREASE TESTS

"•

a few seconds at the junction. Normally there is no sign of a ring. This test
modification of Nonne's Phase I reaction.

A test that is not in general use is strongly recommended by Miller. It is known
as Pandy's test. To carry it out prepare a saturated solution of carbolic acid crystals
in distilled water. Place i c.c. of this reagent in a small test-tube and add i drop
of spinal fluid. In a normal fluid only the faintest opalescence is observed, but in
a fluid with globulin increase a smoke like white cloud developes instantly where
the drop comes in contact with the reagent. Miller gives the following table as
showing the average frequency of the various reactions in syphilis of the central
nervous system.

SHOWING THE AVERAGE FREQUENCY OF THE VARIOUS REACTIONS IN SYPHILIS
THE CENTRAL NERVOUS SYSTEM

Paresis

Tabes dorsalis

Cerebrospinal
syphilis

Blood Wassermann
Spinal fluid Wassermann

98-100%
07%

70%
60-80%

70-80%
8^—00%

Pleocytosis

98%

8q-oo%

8c-Qo%

Positive globulin test.

IOO%

QO~ (K%

OO— Q$%

Colloidal gold test

98-100%

8c-oo%

7<;-8o%

Pare tic
curves

Luetic type
of curve

Luetic
curve

Poliomyelitis in addition to small mononuclear increase together with rather
characteristic large mononuclear cells shows a globulin increase.

Urea Content. — Any excess of urea in the cerebrospinal fluid is a sure
sign of renal inadequacy.

Normally the urea content of cerebrospinal fluid is only 0.006%,
according to Canti. Cases of true uremia, and not renal disease
associated with cardio- vascular disease, show from o.i to 0.6%.
Cases showing over 0.3% rarely recover. The estimate may be made
from 5 c.c. of spinal fluid by the urease method.

CHAPTER XXXIV

RABIES, SMALLPOX, VACCINIA AND THE FILTERABLE

VIRUSES

RABIES is a disease of dogs and wolves, but is communicable to man
and domesticated animals. The virus, whatever it may be, resides in
the saliva and nervous structures. It is destroyed by a temperature of
5o°C. In man the period of incubation is usually from three weeks to
three months, but may be shorter or may extend over one year.

Bites about the face and those with marked lacerations are particularly serious.
Bites of rabid wolves give about four times as great a mortality as those of dogs.
In the dog there are two types of the disease — dumb rabies and furious rabies.

By inoculating rabbits subdurally with an emulsion of the brain or spinal cord
of a rabid animal, and successively the medulla of this rabbit subdurally into other
rabbits, we finally so increase the virulence of the infection that rabbits die in six days.
Beyond this it is impossible to increase the virulence and it is termed fixed virus."
The pathogenic power of this virus is also changed so that it is not apt to cause rabies
if injected subcutaneously. To attenuate this virus the spinal cord of the rabbit
is removed and is dried over caustic potash at a temperature of 23°C. The cord is
divided into segments about i inch in length. Drying for about fifteen days seems
to entirely destroy the virus.

To prepare the material for prophylactic injections a small portion of the cord is
emulsified with normal salt solution and injected subcutaneously. The German
method is to commence with a cord that has been desiccated only eight days. At
first injections are given daily, and it is possible to inject three days' cords by the
sixth day. The immunity is "active" and the immunizing agent is a "vaccine."
Like vaccine virus the product can be preserved (for probably a month) by the use
of glycerine so that it is now possible to send the material for inoculation from the
laboratory preparing it.

The treatment lasts for about twenty days.

There are other methods of treatment as follows:

i. The Harris Method. — In this the brain and cord are ground up
with CO2 snow and the frozen tissue dried over H2SO4. The process
of drying lasts about two days and the virulence of the virus is reduced
one-half. The potency of the virus, when kept at o°C., holds for
six months at least.

429

430

RABIES

iline

2. The Gumming Method. — In this the brain is emulsified in saline
and dialyzed. In this method the virus is so attenuated that injec-
tions do not produce rabies on intracranial inoculation.

3. In the Hogyes method the fresh virulent cord is injected but s
diluted in strength that it acts as does an attenuated virus.

In the diagnosis of rabies in dogs it is preferable to preserve the animal so that
the development of the symptoms may be observed.

In case the dog has been killed, it may be possible to make a diagno-
sis by means of the Negri bodies. These are round or oval bodies from

FIG. 112. — Two nerve cells of hippocampus major (smear preparation) showing
Negri bodies. A, Negri bodies; B, inner bodies within the Negri bodies. (After
Reichel, American Veterinary Review.)

i to 2o/x in diameter, which may be found in the nerve-cells, especially
those of the cornu ammonis (Hippocampus major).

These bodies were first described by Negri in 1903. In street rabies large ame-
boid forms from 18 to 2$n may be found, while in the nerve tissues of animals with
"fixed" virus only minute forms, 0.5/1 or less, may be detected. The fact that the
virus will pass through a Berkefeld filter is no argument against its protozoal
nature. Calkins considers it to be of rhizopod affinity. The name Neuroryctes
hydrophobia has been given it. The bodies are present four to seven days before
the onset of symptoms. They may be demonstrated by staining smears of gray
brain substance by some Romano wsky method, especially by the Giemsa stain.
The smears should be made by mashing the thin slice of gray matter taken from i.
Cornu ammonis, 2. Region of fissure of Rolando — in dog crucial sulcus — or 3. Cere-
bellum, with a cover-glass against the slide. Afterward the cover-glass is gently
drawn along the slide.

The smear on the slide is then fixed in methyl alcohol for two to three minutes,

I

RABIES, SMALLPOX, VACCINIA AND FILTERABLE VIRUSES 431

washed with water and covered with a stain made by adding 3 drops of Sat. ale.
sol. of basic fuchsin to 10 c.c. of distilled water and then adding 2 c.c. of Loffler's
methylene-blue solution. The stain on the slide is then steamed gently and after-
ward washed with water and dried.

As their relation to the nerve-cell is more or less disturbed by such a method
it is preferable to fix brain tissue from the region of the cornu ammonis for five to
seven hours in Zenker's fluid, then to imbed in paraffin and make sections. These
are stained with Giemsa's stain and the Negri bodies are brought out as iliac-red
bodies in the blue cytoplasm of the nerve-cells. It is necessary to differentiate in
95% alcohol.

In the Lentz method the 3;* sections, after removal of the paraffin, are flooded
with absolute alcohol. They are then stained with a ^% solution of eosin in 60%
alcohol for one minute. Wash in water and next stain for one minute in LofHer's
methylene blue. Again wash in water. Apply LugoFs solution to the section for
one minute and then differentiate alternately in methyl alcohol and water until the
section is pink. After washing in water, again stain with LofBer's blue for one-half
a minute, then wash in water and dry carefully with filter-paper. Now differentiate
in alkaline alcohol (i drop of a 5% solution NaOH in 30 c.c. absolute alcohol) until
the section is pink, then quickly differentiate in acid alcohol (i drop 50% acetic
acid in 30 c.c. absolute alcohol) until a slight blue outline to the ganglion cells is
obtained. Treat rapidly with absolute alcohol and xylol and mount in balsam.
The Negri bodies show as light carmine pink bodies on the light blue ground of the
ganglion cells. In the interior of the pink bodies dark blue dots or rings may be
observed.

This method can also be used for brain smears.

In addition to examining for the Negri bodies, a rabbit may be inoculated sub-
durally with a sterile salt-solution emulsion of the medulla of the dead dog.

If the brain and medulla of the dog are to be sent to a laboratory
for examination they should be packed in ice or placed in glycerine.
Take of glycerine one part and one part water. Sterilize the diluted
glycerine by boiling, allow to cool, and drop the pieces of brain tissue
into this. This does not kill the virus.

When from advanced putrefaction, or otherwise, the Negri bodies cannot be
found the changes in the Gasserian ganglia may give a diagnosis. In typical lesions
the ganglion cells are more or less completely destroyed and replaced by cells of
other types.

When a person is bitten by a dog suspected of being rabid the following
simple measures should be instituted. The dog should be kept under observation
in a safe quiet place and will show clinical evidence of rabies within five days and
will die shortly afterward in case rabies exists. When the animal dies the head
and several inches of the neck should be removed and packed in ice and sent to the
nearest laboratory.

Antirabic serum has been prepared by injecting sheep with emulsions of rabbits'
cord and brain— at first intravenously, then subcutaneously.

432 VACCINE VIRUS

The thorough cauterization of the dog-bite wound with pure nitric
acid, as soon as possible after the bite, is imperative even when the Pas-
teur treatment can be given later.

Smallpox. — The etiology of this disease is very obscure, the virus
being grouped under the Chlamydozoa. Smallpox and vaccinia are
often classed as filterable viruses. Park, however, was unable to pass
the virus of vaccinia through a Berkefeld filter with 40 pounds pressure;
the failure may have been due to lack of sufficient dilution. They did
find that the virus would pass through the finest filter-paper.

In 1892 Guarnieri noted cell inclusions in the cornea of rabbits inoculated with
smallpox and under the name Cytorrhyctes variola, Councilman has described what
he regards as a protozoon invading cell nuclei. Certainly ordinary bacteria are not
concerned in the etiology of smallpox. The virus is not only contained in the skin
lesions but also in nasal and buccal secretions, the disease being communicable
before the eruption appears. The period of incubation of variola vera is very con-
stantly twelve days, while that of variola inoculata (a method of prophylaxis by
inoculating discharges from a vesicle or pustule which preceded the present
method of vaccination) is eight days. Monkeys are quite susceptible to both small-
pox and vaccinia as is also true of corneal inoculations of rabbits.

Cutaneously the rabbit shows a typical eruption after vaccination,
but does not show characteristic lesions after smallpox inoculation.

It is usually accepted that vaccinia is simply a permanently modified smallpox
resulting from animal passage and it is stated that repeated passage of smallpox
virus through calves produces vaccine virus. Inoculation of calves with smallpox
virus is a most uncertain procedure and Park states that he has been unable to obtain
success after many such experiments.

Of great practical importance is the differentiation of smallpox
and chicken-pox. In efficiently vaccinated persons the cutaneous in-
oculation of material from the suspicious vesicle will give rise to a skin
reaction similar to the Pirquet Tb. one, occurring within twenty-four
hours. If the vesicle were of chicken-pox no reaction occurs. Heating
the material to 6o°C. for thirty minutes eliminates all danger and does
not interfere with the reaction. (Tieche.)

Vaccinia. — Vaccinia is a disease produced artificially by the injection
of vaccine virus obtained from the calf. The material for vaccine is
taken from vesicles about one week after the inoculation. The most
potent material is in the pulp at the base of the vesicle and not in the
lymph which exudes from the vesicle. The pulp is ground up and mixed
with an equal amount of glycerine, which acts not only as a preservative
but as a mild antiseptic for nonsporing bacteria. The calves are autop-

RABIES, SMALLPOX, VACCINIA AND FILTERABLE VIRUSES 433

sied after the pulp has been curetted from the inoculated skin of the
abdomen to be sure that no disease exists in the calves. The virus is
afterward tested for pus organisms, tetanus, and foot and mouth
disease.

Very important is the test for tetanus. Cultures are grown anaerobically for six
days, then filtered and the filtrate inoculated into guinea-pigs and these latter
watched for ten days to note evidence of tetanus.

If found free from any harmful germs the vaccine is then tested upon children for
potency. Incision is the proper method of vaccination, as by scratching with a
needle, two lines i inch long and i inch apart. Scarification should never be
practised.

Guarnieri in 1892 first observed small bodies near the nucleus of infected epi-
thelial cells. He called them Cytoryctes vaccinia. Calkins regards these bodies
as well as the Negri bodies as being rhizopods and the distributed chromatin as
idiochromidia (granules of nuclear chromatin within the cytoplasm).

THE FILTERABLE VIRUSES

The first disease of which the virus was found to be capable of pass-
ing through the finest porcelain filter was that of foot and mouth
disease (Loffler and Frosch, 1898).

The filter which is ordinarily used for testing for the passage of such
disease agents is the Berkefeld filter, one made of diatomaceous earth.
The filter should be new and sterilized before use. The material
should be diluted with saline before filtering. One may use slight
suction from a filter pump. The filtration should occupy only a short
time, not exceeding two hours.

Of the infections belonging to man, in which such a passage of blood
or serum through the pores of a porcelain filter, capable of keeping
back even such a small bacterial organism as that of Malta fever, but
which does not hold back their virus, we have the following: foot and
mouth disease, trachoma, molluscum contagiosum, vaccinia, variola,
rabies, typhus fever, measles, scarlet fever, yellow fever, dengue,
Papataci fever, poliomyelitis and coryza. It has been suggested that
the virus of cerebrospinal meningitis may be a filterable one.

Hog Cholera Virus. — Very interesting is the history of the virus of hog cholera or
swine fever. This was supposed to be due to an organism of the hog cholera group,
B. aertrycke (identical with B. cholera suis and B. suipestifer). This organism
belongs to the "enteritidis" group and is more common as a cause of food poisoning
in man than the better known Gaertner bacillus. Recently the group of organ-
isms, including the paratyphoid B., but not A, as well as the hog cholera group,
28

434

FILTERABLE VIRUSES

has been designated the Salmonella group. It is now known that the cause of
most important fatal disease of swine is a filterable virus.

This virus shows remarkable powers of resistance to external influences, thus it
can be kept for months in animal tissues. It is not destroyed by drying and with-
stands a temperature of 58°C. for two hours but not one of 72°C. for one hour.
Cell inclusions have been found in smears from the conjunctivas of hogs sick with
the disease. See Chlamydozoa. A very valuable prophylactic but not curative
serum is found in the serum of animals recovering from the disease or in those
immunized.

There are many other diseases of this nature which are important among the
domesticated animals, such as pleuropneumonia of cattle, African horse sickness
and hog cholera. The viruses of pleuropneumonia of cattle and poliomyelitis have
been obtained in artificial cultures. Some of these viruses seem related to bacterial
infections and others to protozoal ones. These viruses differ as to method of trans-
mission, pleuropneumonia of cattle being transmitted by inhalation, rabies and
vaccinia by the cutaneous atrium, hog cholera by ingestion and many of those sup-
posed to have protozoal affinities, as yellow fever, Papataci fever and horse sickness
by mosquitoes.

As a rule these viruses are destroyed by a temperature of 55°C. in a few minutes.

CHAPTER XXXV

DISEASES OF UNKNOWN OR NOT DEFINITELY DETERMINED

ETIOLOGY

OF TEMPERATE CLIMATES

Acute Articular Rheumatism. — Various bacteria have been reported
as cause. The organism which seems the most probable cause is the
short chain coccus, Micrococcus rheumaticus of Triboulet and others.
Inoculations of this streptococcus cause polyarthritis and pericarditis.
Poynton and Paine have cultivated the organism from the cerebro-
spinal fluid in three cases where chorea was present.

The Common Cold. — Of all the diseases common in man this condi-
tion has been surrounded by greater etiological and epidemiological
obscurity than any other.

We are inclined to believe that the common cold (coryza) sets in when our re-
sistance is lowered by alimentary tract disturbances, from exposure to variations
in temperature, or following refrigeration and fatigue. Of course, many have held
that the common cold was "catching" but the evidence offered in support of such
a view has been academic.

Many bacterial organisms have been suggested as causative such as
B. coryza segmentosus, haemolytic and viridans types of streptococci,
M. catarrhalis, etc. In 1914 Kruse brought forward evidence to prove
that the etiological factor in coryza was a filterable virus. Quite re-
cently Foster has conducted experiments in which, by using the nasal
discharge from typical coryza cases, diluting it with 10 or 15 times its
volume of saline, then passing through a small Berkefeld filter and in-
stilling 3 to 6 drops of the filtrate into the nasal cavity of 10 well men
he produced typical coryza in nine of these men in from eight to thirty
hours. Cultures were made from the filtrate following Noguchi's
spirocha?te culturing method. The culture medium surrounding the
piece of sterile tissue showed turbidity in from forty-eight to seventy-
two hours and dark-field examination showed myriads of extremely
active bodies which were thought to possess true motility rather than
Brownian movement.

435

436 POLIOMYELITIS

Filtrates from these cultures were instilled into the nasal cavity of n men and
after a period of incubation of from eight to forty-eight hours all came down
with coryza.

Epidemic Poliomyelitis. — Material from the cord of child with the
disease when injected subdurally, intravascularly, or into the peritoneal
cavity of monkeys produced the disease in the animals inoculated.
The virus has been passed through three generations of monkeys
(Flexner).

The virus has been found in the brain, spinal cord, mesenteric and salivary glands
of monkeys and may remain in the nasal mucosa of monkeys as long as five months.
This would indicate the existence of human chronic carriers. With the possible
exception of the rabbit only man and the monkey are susceptible. This would
indicate that the virus is directly transferred from man to man. The virus is
highly resistant to drying and light. It will remain alive for months in dust. It is
not sterilized by pure glycerine during many months of contact. It is possibly
transmitted by a biting fly, Stomoxys calcitrans. Against this is the fact that
the virus has not been found in the blood.

Flexner and Noguchi have recently cultivated the virus of polio-
myelitis by employing ascitic fluid to which had been added a fragment
of sterile rabbit kidney and nutrient agar, this culture medium being
covered with a layer of paraffin oil. The growth is obtained under
anaerobic conditions. The minute colonies are composed of globular
or globoid bodies from 0.15 to 0.3 micron in diameter. These bodies
may be single or in chains or in masses. In older cultures bizarre forms
are obtained. Monkeys have been inoculated with the cultures.

Foot and Mouth Disease. — Due to an ultramicroscopic organism.

This is a highly contagious disease of cattle characterized by the appearance
of vesicles in the mouth and about the feet of cattle. Man rarely contracts the
infection through drinking the milk of infected animals. This disease is of great
interest as having been the first of the filterable virus diseases to have been discovered
(Loffler and Frosch in 1898).

Measles. — Cause entirely unknown. Hektoen has shown that bl
contains the virus.

Anderson has found that the virus of measles can pass through a Berkefeld filter
and loses its infectivity after heating for fifteen minutes at 55°C. In infecting
monkeys it was found that the blood of patients with measles was infective only
just before and for about twenty-four hours after the appearance of the eruption.
Mixed nasal and buccal secretions were infective for monkeys for about forty-eight
hours from the time of the eruption. The scales from desquamating cases were
not capable of infecting monkeys hence it was thought that measles was no
contagious during the period of desquamation.

DISEASES OF UNKNOWN ETIOLOGY 437

Mumps. — Herb has implicated a diplococcus. Inoculations into
Stenson's duct of monkeys successful.

Rabies. — Probably the Negri bodies.

Roetheln (German Measles). — Nothing known.

Scarlet Fever. — Streptococci seem most probable cause (S. anginosus).
Mallory has implicated epithelial protozoa.

Dohle has reported the rather constant finding of basophilic round or oval inclu-
sion bodies in the polymorphonuclears. These findings, which are brought out in
stained films, are present only during the first few days of the attack of scarlatina.
Other workers have found these bodies almost as constantly present in diphtheria
as in scarlet fever. They have also been found in other acute diseases than diph-
theria. Klemenko has obtained streptococci from the blood in only about 2% of
cases of scarlatina. Quite recently Mallory has reported diphtheroid organisms
as the cause.

Smallpox and Vaccinia. — Guarnieri and Councilman have impli-
cated epithelial protozoa.

Spotted Fever of the Rocky Mountains. — Supposed to be due to an
unknown protozoon transmitted by a tick, D. andersoni.

This disease is especially prevalent in the Bitter Root Valley of Montana and
to some extent in the mountains of Idaho. It is an acute febrile affection with a
tendency to stupor. The eruption, which appears about the third to fifth day, is
not unlike that of typhus fever and tends to become haemorrhagic. Gangrene oi
penis or scrotum may appear. It is transmitted by a tick, Dermacentor andersoni
(D. venustus) which lives on domesticated animals of the region. Destruction of
ticks which attach themselves to sheep by dipping has been proposed as a measure
for eradication of the disease. Ricketts found that the reservoir for the virus is
to be found in ground squirrels, chipmunks, mountain rats, etc., and that ticks
feeding upon them become infected and so transfer the disease to man. Guinea-
pigs are susceptible as is also the monkey. Ricketts noted certain chromatin staining
bacteria in man and in eggs of infected ticks as possibly playing a part in etiology.
Quite recently Wolbach has reported the finding in infected guinea-pigs of organ-
isms, possibly bacterial, showing granular and lanceolate forms. They are par-
ticularly abundant in the endothelial cells of blood-vessels. They are from ^ to
i micron long by about J^ micron broad. Ricketts stated that his organisms were
about the size of B. influenza and showed as two lanceolate-shaped bodies. Wilson
and Chowning, in 1902, reported the finding of piroplasm-like organisms in the
blood of the disease. Ricketts proved that the virus was not filterable.

Trachoma. — This contagious form of granular conjunctivitis is
supposed to be due to chlamydozoa or inclusion bodies and is classed
as one of the filterable viruses. The relation of the trachoma bodies
to the Koch- Weeks bacillus is discussed under that organism.

438 BERIBERI

Typhus Fever. — It has been suggested that the cause may be a pro-
tozoon transmitted by vermin.

Recent work by Anderson and Ricketts has shown that the blood of
human cases is infective for monkeys. The virus does not seem to
pass through a Berkefeld filter and the epidemiology points to the
body louse as the transmitting agent. Nicolle reported the filter-
ability of the virus.

Plotz has isolated a Gram-positive pleomorphic bacillus from the blood of typhus
patients as well as from the blood of guinea-pigs and monkeys infected by injections
of typhus blood. It is most abundant in blood taken four or five days before the
crisis. It only grows anaerobically and grows best in ascitic fluid sterile tissue media.
Morphologically it shows curved, straight and coccoid forms. The rods are about 1.5
micron long. The serum of convalescents shows complement-fixation bodies as
well as agglutinins. The organism has been named B. typhi exanthematici.

Hort states that only blood recently taken from typhus patients will cause the
disease in monkeys while the same blood which has been incubated several hours
or days fails to produce the disease. Others, as well as Hort, doubt the etiologi-
cal relation of the organism of Plotz to typhus fever or the mild form of the
disease as seen in New York City and there known as Brill's disease. Tabardillo
or Mexican typhus is the same as typhus.

Varicella. — Entirely unknown.

Whooping-cough. — Influenza-like bacilli have been implicated.
Bordet-Gengou bacillus.

OF TROPICAL CLIMATES

Ainhum. — A disease characterized by a constricting fibrous ring,
especially of little toe, often leading to spontaneous amputation.

Beriberi. — Various microorganisms and food factors suggested.
A form of multiple neuritis, occurring chiefly in countries where rice
is the staple food, characterized by oedema and marked cardiac and
respiratory embarrassment. The vagal involvement produces grave
symptoms. Rice from which the pericarp has been largely removed,
polished rice, implicated.

Prior to the investigations of Fraser and Stanton the importance of the rice factor
in the etiology of beriberi was insisted upon by Braddon who thought that a poison
was elaborated by some organism which poison was contained in the beriberi pro-
ducing rice. This development was thought to occur in rice stored in damp places,
but Vedder has shown that storing undermilled rice in a damp place for a year does
not cause it to lose its anti-beriberi producing properties. The work of Eijkman in
showing that polyneuritis could be produced in fowls by feeding them on polished
rice and prevented when a diet of rice polishings was added to the neutritis-produc-

DISEASES OF UNKNOWN ETIOLOGY 439

ing rice opened the way for a vast amount of experimental work. As regards the
nature of the neuritis preventing substance in the rice polishings it was soon found
that it had no relation to the phosphorus content. Funk has isolated a substance
he calls vitamine, a pyramidine base precipitated by phosphotungstic acid, which is
present in rice in the proportion of i to 100,000 and seems to possess extraordinary
curative properties in polyneuritis gallinarum. Heart muscle, egg yolk and yeast
are rich in this anti-neuritis substance, which is also present in lentils and barley.
Schaumann considers malt as richer in the anti-neuritis vitamine than any other
article of diet, rice bran coming next. Many think that vitamines have not as yet
been separated but that they are intimately combined with some mother substance
in the food. There is, in all probability, a large number of vitamines present in
various animal and vegetable foods, the deficiency of which in a diet may lead to
vague disorders or to well-recognized diseases, such as scurvy, ship-beriberi, beriberi
or pellagra.

Schaumann considers the curative principle to be of the nature of
an activator. An increase in the ingestion of carbohydrates and nec-
essarily in the vitamine as well seems to produce neuritis more rapidly
than where a smaller amount is given, this indicating the importance
of these vitamines in carbohydrate metabolism.

In epidemics of beriberi it has been observed that those who eat most rice are more
often attacked, thus men more frequently than women. A temperature of 1 2o°C. de-
stroys the vitamine. Owing to the absence of rice as a constituent of other than slight-
est importance in the dietary of Brazilian cases of beriberi, as well as from numerous
reports of the occurrence of the disease in nonrice-eating persons, the view that is
now entertained is that not only polished rice, but any predominating carbohydrate
article of diet, which is deficient in the neuritis-preventing substance, can produce
beriberi. Wellman and Bass have shown that such articles of diet as sago, boiled
white potatoes, corn grits and macaroni practically parallel polished rice in the pro-
duction of polyneuritis in fowls.

Blackwater Fever. — Considered as a malarial disease, but thought by
some to be possibly caused by a protozoon — a Babesia (Piro plasma).
A disease usually occurring in patients with a malarial history and
characterized by rapid febrile onset, early jaundice, asthenia, pain in
loins and the pathognomonic haemoglobinuria.

Dengue. — Supposed to be due to a protozoon transmitted by Culex
fatigans. A disease characterized by sudden onset, high fever for three
or four days, pains in the postorbital regions, back and about joints.
A remission occurs on the third to fifth day followed by a secondary
rise of temperature and a measles-like eruption. Leukopenia and re-
duction in the percentage of polymorphonuclears. Virus exists in
the blood and is filterable.

440 PELLAGRA

Goundou. — Symmetrical bony tumors of nasal processes of superior
maxillary bones.

Oroya Fever. — A disease with a fever characterized by a profound
involvement of the bone marrow producing very rapidly an anaemia
resembling that of pernicious anaemia. Pains of bones and joints
marked. See Verruga.

The disease is chiefly found in towns situated in narrow, wind-protected valleys
of the West side of the Andes, at elevations of from 3000 to 9000 feet. Townsend
has suggested that a species of Phlebotomus, which is very prevalent, may be the
transmitting agent.

Barton isolated a paratyphoid bacillus from the blood of a patient, besides which
other bacteria have also been isolated. In 1909, Barton noted certain rod-like
organisms in the red cells of Oroya fever patients which he considered protozoal in
nature.

Strong and his colleagues found in the blood of Oroya fever cases
rod-shaped forms in the red cells, varying from i to 2 microns in length,
the red cells containing from i to 30 of these elements.

Intravenous inoculation of blood containing these elements into monkeys and
rabbits was negative in result. These organisms were considered as intermediate
between bacteria and protozoa. They are closely related to Grahamella and the
Harvard commission has proposed the name Bartondla bacilliformis.

Pellagra — This disease is characterized by (i) a sprue-like stomatitis
and disorders of alimentary canal, (2) an erythema usually limited to
parts exposed to the sun and characterized by marked symmetry and
striking delimitation from the sound skin and (3) various neurological
manifestations and a toxic psychosis which may go on to confusional
insanity. The disease is characterized by annual recurrences in the
spring with improvement in the winter. The views as to etiology
which consider the causative agent as a protozoon, possibly transmitted
by a Simulium or by Stomoxys are now historical.

The maize ideas of etiology are now considered as having a bearing only in con-
nection with the larger question of vitamine deficiency in cereals in general. Aless-
andrini has recently brought forward the colloidal silica etiology.

These views are that colloidal silica in water is responsible for the disease. Voegt-
lin noted the great amount of aluminium in certain vegetables and suggested this
as the toxic causative substance. A mixture of colloidal alumina and silica in water
is supposed to be operative as well as silica alone. Against the colloidal silica
hypothesis is the statement of Sandwith that the water of the Nile, the drinking
water of Egypt, is low in colloidal silica content.

DISEASES OF UNKNOWN ETIOLOGY 441

The present trend of thought in connection with pellagra is that it
is a food deficiency disease connected with a deficiency in vitamines
necessary for normal metabolism (see beriberi).

In February, 1915, Goldberger started a "pellagra squad," consisting of n
prisoners on a diet of wheat flour (patent), cornmeal, corn grits, corn starch, polished
rice, granulated sugar, cane syrup, sweet potatoes, fat fried out of salt pork, cabbage,
collards, turnip greens and coffee. Baking powder was used for making biscuits
and corn bread. The food value of each man's diet averaged 2952 calories.

A control was carried out with prisoners on a normal diet. The experiment was
continued until Oct. 31, 1915. Of the n volunteers on the excessive carbohy-
drate diet six developed symptoms. Loss of weight and strength and mild neuras-
thenia were early symptoms. Definite cutaneous manifestations appeared only
after five months. The skin lesions were first noted on the scrotum, later appearing
on backs of hands in two cases and back of neck in one case.

Just as with rice so does excessive milling of wheat get rid of vitamines, therefore
bread made from highly milled flour is dietetically deficient.

Again, as brought out by Voegtlin, alkalis tend to destroy any remaining vitamines
in such bread. The practice of using sodium bicarbonate in preparation of bread
is a further factor in the food deficiency problem. With the use of baking powder
or buttermilk the alkaline carbonate of soda is neutralized so that there is no de-
structive effect on vitamine content.

Notwithstanding the above evidence as to food deficiency etiology it must be re-
membered that McNeal and his colleagues on the Thompson-McFadden pellagra
commission, as well as other authorities on this disease, insist upon a probable
infectious agent as cause.

Rat-bite Disease. — A disease caused by the bite of rats. Rather
common in Japan. Five weeks after bite when wound has healed,
high fever sets in, cicatrix becomes inflamed with lymphangitis and
swollen glands. The fever falls in a few days to be succeeded by other
febrile paroxysms. An erythematous eruption accompanies the second
paroxysm.

Supposed by Ogata to be due to a protozoon, but recent work by Schotmuller,
in 1914, has shown that the cause is a Streptothrix, S. muris ratti. This finding has
been corroborated by Blake. The organism first invades the lymphatic structures
and then the blood, giving a septicaemia. Various organs are later involved. Blake's
case developed a powerful agglutinin for the specific Streptothrix.

Sprue. — A form of chronic diarrhoea characterized by diaphanous
thinning of gut and ulcerations of buccal cavity.

Kohlbrugge found organisms resembling Oidium albicans in the intestines,
oesophagus and tongue. He found similar organisms in the stools and tongue
scrapings of cases of sprue. Beneke found bacilli in the tongue, oesophagus and
intestines and considered these as causative, regarding the thrush-like membranous
deposit as connected with the cachectic state and not causative.

442 YELLOW FEVER

Bahr is inclined to believe that Monilia albicans (Oidium albicans} is the cause
as he found these saccharomycetes in the deep layers of the tongue, in the mucoid
coating of the intestines and in the deposit in the oesophagus. He thinks it the
ordinary thrush species which may take on greater virulence in the tropics. Ashford
states that he has found a species of Monilia, different from that of thrush, almost
constantly in tongue scrapings and stools of sprue cases and he regards this species
as the cause of sprue. He states that this organism is common in Porto Rico bread
and thinks it possible that the disease is transmitted in this way. Wood has re-
cently expressed the view that sprue is not infrequently mistaken for pellagra in
the southern United States.

Tsutsugamushi. — A disease of Japan somewhat resembling typhus
fever. Supposed to be due to a protozoon transmitted by the Kedani
mite.

Verruga Peruana. — A disease of Peru formerly considered as a later
stage of Oroya fever.

The eruption of verruga somewhat resembles that of yaws and it was at one time
suggested that verruga was simply yaws as influenced by high altitude. Strong
and his colleagues found that they could infect rabbits intratesticularly and that
lesions resembling those of man could be produced in dogs and monkeys by cuta-
neous and subcutaneous inoculations. The virus has been transmitted from monkey
to monkey. The Wassermann reaction was negative. In extracts from the granu-
lomatous lesions they found a very active haemolysin. It will be remembered that
animals are not susceptible to Oroya fever blood inoculations.

From the fact that it is possible to inoculate a person by rubbing verruga material
on a scarified surface it would seem that the infection might be transmitted by
insects.

Yellow Fever. — Due to a filterable virus transmitted by the Stego-
myia calopus. A disease characterized by sudden onset, rachialgia,
albuminuria, jaundice and often haemorrhages about the third day.
Pulse becomes slow even with rising temperature. Black vomit often

precedes fatal termination. Virus exists in the blood and is filterable.
P

The virus is present in the blood of the peripheral circulation only during the
first three days of the disease. A female Stegomyia sucking the human blood
during the first three days from onset of fever may become infected but cannot
transmit yellow fever to a second person until after the expiration of at least twelve
days, during which time some development of the virus, of the character of which
we are in ignorance, goes on in the mosquito.

Seidelin has stated that he has found a protozoon, Paraplasma
flavigenum, in the blood of yellow-fever patients. Authorities generally
deny the existence of this parasite.

APPENDIX

A— PREPARATION OF TISSUES FOR EXAMINATION IN
MICROSCOPIC SECTIONS

Note. The most important step in the preparation of sections of tissues for histological
examination is proper and immediate fixation. This step in the technic is often
in the hands of the surgeon at the time of the operation or the physician at autopsy
and it should be understood that a satisfactory diagnosis can only be made when
the pieces of tissue are at once dropped into a fixative. Various protozoa, as amoebae,
disintegrate in one or two hours, unless properly fixed and body cells show degen-
eration after the tissues have been left without fixation for a few hours, which
changes may be interpreted as pathological.

Prepare a pint or quart of 10% formalin solution (4% formaldehyde) shortly
before operation or autopsy. Drop into the solution slices of tissue, not more than
*y± inch thick, as soon as cut. Leave in the fixative for twenty-four hours and
then place in 70% alcohol. The pathologist will attend to the other steps.

We use two fixation solutions in routine work, one of 10% formalin and one of
Zenker's solution. This latter requires prolonged washing of tissues following
fixation and has little advantage over formalin for ordinary purposes.

i. Fixation:

a. It is most important that the tissues to be examined be placed in the fixing
fluid as soon after death or operation as possible. Degenerative changes are in this
way avoided.

b. The piece of tissue to be fixed must not be too large. Using a sharp scalpel,
or preferably a razor, a slab of tissue about one-half an inch square and not more
than one-fifth of an inch thick should be dropped into the bottle containing the fixa-
tive. The bottom of this bottle should have a thin layer of cotton with a piece of
filter-paper covering it. There should be at least 20 times as great a volume
of fixing fluid as of tissue to be fixed. Delicate tissues, as pieces of gut, should be
attached to pieces of glass, wood, cardboard, or blotting paper before being placed in
the fixative.

c. The most convenient fixative for the average medical man is (i) a 10% solution
of ordinary commercial formalin (4% of formic aldehyde gas), either in water or,
preferably, in normal salt solution. Fixation is complete in from twelve to twenty-
four hours. By placing in the incubator, at 37°C., two to twelve hours in the forma-
lin solution suffices. If fixed in the paraffin oven (s6°C.), fixation is accomplished
in about one-half hour.

Formalin once used for fixation must be thrown away.

(2) The fixative which probably gives the best histological pictures and with which
we obtain the most satisfactory haematoxylin staining is Zenker's fluid. This is

443

444 APPENDIX

Miiller's fluid containing 5% of corrosive sublimate. It also contains 5% of glacial
acetic acid, which latter is only added just before we are ready to fix the piece of
tissue. Muller's fluid is:

Pot. bichromate, 2 . 5 grams.

Sod. sulphate, i . o grams.

Water, loo.oc.c.

Zenker's fluid fixes in about twenty-four hours. After all corrosive sublima
fixatives we should wash the tissues in running water for twelve to twenty-four
hours. The precipitate of mercury in the tissues is best gotten rid of by treating the
section on the slide with Lugol's solution, rather than the tissue in bulk with iodine
alcohol.

(3) In Orth's fluid we add 10% of formalin to Muller's fluid (recommended for
nerve tissue).

A saturated corrosive sublimate solution in salt solution with the addition of 5%
of glacial acetic acid may be used as a substitute for Zenker's fluid.

(4) Where the tissue is to be examined chiefly for bacteria absolute alcohol is the
best fixative. The piece of tissue should be small, not over ^ inch thick and sus-
pended by a string to the cork so as not to lie on the bottom where the alcoholic
strength tends to become weaker. Better histological details are gotten by fixing
for two hours with 80% alcohol and then transferring to absolute for twelve to
twenty-four hours.

2. Dehydration. — After washing for twelve to twenty-four hours in running
water, following corrosive sublimate fixation, or simply washing for a few minutes
after formalin, the tissues should be placed in 70% alcohol. They may be kept in
this indefinitely. If they are to be sent to a laboratory for sectioning, it is advisable
to moisten a pledget of cotton in 70% alcohol and fill in the bottom of the bottle
with it. Then drop in the tissues and pack in gently over them sufficient 70%
alcohol-saturated cotton to fill up the bottle. All the alcohol should be absorbed
by the cotton so that if the bottle should break in transit there would be no damage
from the alcohol. The stopper of the bottle should be paraffined or sealed with
wax.

Tissues may be left in the 70% alcohol twelve to twenty-four hours and should
then be transferred to 95% alcohol for an equal time. They are then transferred to
absolute alcohol, where they remain from two to twelve hours and are then placed in
xylol. The time in xylol should be as short as possible. So soon as the tissue looks
clear it should be removed — thirty minutes to two hours.

Bolles Lee is a strong advocate of the superiority of cedar oil over xylol or any other
clearing agent for paraffin imbedding. It does not affect delicate structures nor make
them brittle even when kept in the cedar oil for weeks or months. Furthermore,
it does not matter whether the cedar oil is entirely gotten rid of before sectioning the
paraffin as is the case for best results with xylol. Cedar oil will clear from 95%
alcohol as well as from absolute alcohol.

3. Imbedding. — The tissue is now transferred to melted paraffin. Paraffin melt-
ing at 48°C. for winter work, and that melting at 54°C. for summer is to be recom-
mended. The time in the paraffin should not be prolonged. Two hours will
ordinarily suffice. Some leave in the paraffin for twelve to twenty-four hours.

APPENDIX 445

Next take a paper box (made of stiff writing-paper folded over a square of wood)
and fill with the melted paraffin. As quickly as possible drop in the piece of tissue
taken out of the paraffin bath with heated forceps and, so soon as the paraffin begins
to solidify on the surface, place the paper box in ice water. When paraffin is rapidly
cooled, crystallization is less.

The Acetone Method. — Take the tissues out of the 70% alcohol and place in ace-
tone. After remaining in acetone for one or two hours, the tissues should be trans-
ferred to fresh acetone for an equal length of time. Dry calcium chloride in the bot-
tom of the acetone bottles keeps it dehydrated. They should then be placed in
xylol for about one-half hour and then embedded in paraffin as directed above.

The Chloroform Method. — The procedure may be the same as in the method of
passing through alcohols to xylol, substituting chloroform for xylol and then trans-
ferring to paraffin.

Where absolute alcohol is not obtainable, very satisfactory results may be
obtained by transferring tp a mixture of 95% alcohol and chloroform after immer-
sion in 95% alcohol. Then going from the alcohol-chloroform mixture to pure
chloroform, thence to paraffin.

Rapid paraffin imbedding methods.

When a piece of tissue is not more than Y± inch square and ^ inch thick, it is
very easy to run it through in three to six hours. Thus:

10% Formalin (in 37°C. incubator), i hour.

70% Alcohol (in 37°C. incubator), i hour.

95% Alcohol (in 37°C. incubator), i hour.

Absolute alcohol (in 37°C. incubator), H hour.

Xylol (in 37°C. incubator), V?. hour.

Paraffin (in 55°C. incubator), ^ to 2 hours.

Method of Lubarsch. — In this excellent method small pieces of tissue not more
than }/$ inch thick are placed in a wide test-tube containing 10% formalin for 10 to
15 minutes, changing the fluid twice. Transfer to 95% alcohol 10 minutes changing
alcohol once. Absolute alcohol, for 10 minutes changing twice. Pure aniline oil
until tissues are transparent, 15 to 30 minutes. Xylol, changing two to three times
or until the xylol is no longer yellow, 10 to 20 minutes. Imbed in paraffin for 20
minutes to i hour. During the entire process keep the test-tube in a water bath
or incubator at 5o°C. It is necessary to have a good microtome. The best is that
of Minot. Very satisfactory sections can be cut with the various types of student
microtomes, costing from twelve to twenty dollars.

(In using a hand microtome, a razor with a flat edge is necessary. After expe-
rience, sections thin enough for histological but not for bacteriological examination
can be made.)

If the piece of tissue is properly dehydrated and imbedded, thin sections (3 to
ioju) should be easily obtained, provided the knife be sharp. One advantage about
the paraffin method is that it is only necessary to have a small part of the blade in
proper condition. With celloidin the entire cutting edge must be perfect. Having
cut the sections, they should be dropped on the surface of a bowl of warm water
(45°C.). This causes the section to flatten out evenly.

Decakification.—This is best accomplished by fixing in 10% formalin for twenty-

446 APPENDIX

:h square

four hours, then placing a small piece of the bone (not exceeding ^ inch sqi
and % inch thick) in concentrated sulphurous acid.

This decalcifies in about two to seven days. Wash thoroughly in alkaline water
and then in tap water. Pass through alcohols and xylol and imbed and section as
before described.

To Stain Sections. — It is first necessary to affix the section to a slide or cover-glass

To attach the section firmly to the slide, so that it will not become detached in
subsequent treatment, pick up a section on a strip of cigarette paper.

A sheet of cigarette paper is cut into about five pieces (% X iM inch). In-
serting the strip of cigarette paper under the section, it is easily lifted up out of
the water. Then apply the slip of cigarette paper, section downward, to a perfectly
clean slide. Blot with a piece of filter-paper, then strip off the piece of filter-paper
leaving the section smoothly applied to the slide. Next place in the 37°C. incubator
for twelve to twenty-four hours and the section will be found to be so firmly attached
that it will not be dislodged by subsequent treatment.

For Immediate Diagnosis. — Take a very small loopful of albumin fixative (white
of fresh egg, 50 c.c., glycerine, 50 c.c.; sodium salicylate, i gram) and deposit it on a
cover-glass. Now take up a loopful of 30% alcohol (i drop of 95% alcohol and 2
drops of water) and applying it over the albumin fixative, smear out the mixture
uniformly over the cover-glass.

2. Pick up a section on a strip of cigarette paper and apply it to the prepared sur-
face on the cover-glass. Blot with gentle pressure with a piece of filter-paper over
the strip of cigarette paper, and strip off this latter, leaving the section attached to
the cover-glass.

3. Now, turning the flame of the Bunsen burner down very low or with a small
alcohol flame, we hold the cover-glass in a Stewart's forceps, section side up, over
the flame and slowly lower it until the paraffin is observed to melt. This shows a
temperature of about 5o°C. The section is fixed by the coagulation of the albumin
at about 7o°C. To obtain this temperature lower the cover-glass still more, and the
moment vapor is seen to rise from the section it indicates the attachment of the sec-
tion to the cover-glass.

4. Flood section on cover-glass or slide with xylol; this dissolves out the paraffin.
It is better to pour off the first xylol and drop on fresh xylol (one minute).

5. Remove xylol with two applications of absolute alcohol (one minute).

6. Treat specimen with two or three applications of 95% alcohol (one to two
minutes).

7. Next wash in water (one to two minutes).

8. Flood specimen with haemalum or Delafield's haematoxylin (three to seven
minutes).

9. Wash in tap water for about two to five minutes until a purplish tinge is devel-
oped hi the section. The alkali in ordinary tap water develops this color.

10. Apply i to 1000 eosin (aqueous) for thirty seconds to one minute.

11. Wash in water; then in 95% alcohol; then in absolute alcohol.

12. Apply a few drops of xylol and as soon as the section is perfectly transpa
mount in balsam, or immersion oil.

The staining by haematoxylin and eosin is the best for the study of the histology
of a section. It only requires about ten minutes to run a preparation through for
diagnosis by this method.

APPENDIX 447

The reagents are best kept in dropping-bottles.

The staining of sections on slides is exactly as for those on cover-glasses. Cop-
lin's staining jars are very convenient for use in staining slides.

Where the cover-glass method is used, staining by Gram's method, acid-fast stain-
ing, capsule staining, etc., may be carried out as for bacterial preparations.

For staining Gram-positive bacteria in sections, the Gram method as for bacterial
preparations, using dilute carbol fuchsin as a counterstain, gives good results.

For Gram-negative bacteria stain with thionin as for blood preparations (ten to
twenty minutes). Then differentiate in i to 500 acetic acid solution for ten to
twenty seconds, wash with water, then with 95% alcohol, and quickly through abso-
lute alcohol and xylol.

Nicolle's Method.— i. Stain with Loffler's methylene blue ten to fifteen minutes.

2. Differentiate in i to 500 acetic acid ten to twenty seconds.

3. Place in i% solution of tannin for a few seconds (fixes color).

4. Wash in water, then into 95% alcohol, absolute alcohol, xylol, and balsam.

WEIGERT'S IRON ILEMATOXYLIN

Solution I

Haematoxylin, i gram.

Alcohol (95%), 100 c.c.

This must be allowed to ripen for some days and does not keep over six months.

Solution H

Liq. Ferri sesquichlor. sp. gr. 1.124 (about 10%) 4 c.c.

HCL, i c.c.

Water, 100 c.c.

Mix equal parts of number one and number two. The mixture only keeps about
three days. The HC1 prevents overstaining.

This stain followed by Van Giesen's stain gives more perfect results than any
common method of staining. The iron haematoxylin intensifies the sharpness of
the Van Geisen differentiation.

Other iron haematoxylin stains are given under staining for protozoa.

Van Giesen's Stain. — Take of i% aqueous solution acid fuchsin from 5 to 15
c.c. Saturated aqueous solution picric acid 100 c.c. The method of using is to first
stain with haematoxylin in the usual way. Then pour on the picric-acid fuchsin
solution and allow to stain for one to five minutes. Wash, pass through alcohols
and xylol and mount in balsam.

Connective-tissue fibers, axis cylinders, and ganglion cells are stained a bright
garnet red. Myelin, muscle fibers, and cells generally are stained yellow. Nuclear
staining is that of haematoxylin. The stronger stain is used for nerve tissue; the
weaker, for demonstrating connective tissue in tumors.

Levaditi's Method. — Take small pieces of tissue, about 2 mm. in thickness, and
harden in 10% formalin for twenty-four hours and then in alcohol for the same pe-
riod; then wash in water for a short period. They are stained in a freshly made solu-
tion of silver nitrate 1.5% for three successive days, changing the solution each day,

448 APPENDIX

maintaining the blood temperature, and excluding light. The tissue is then ph
in a 2% solution of pyrogallic acid, with the addition of 5% formalin. After remain-
ing in this for twenty-four hours, light being excluded, they are passed through
85%, 95%, and absolute alcohol, respectively; embedded in paraffin; and cut in
about 5 micron sections. Equally good results may be obtained by allowing the
silver nitrate to act at room temperature and embedding in celloidin.

Nogitchi has used the following modification in demonstrating spirochaetes in
brain and cord: Fix ^-inch slabs of tissue in 10% formalin for four or five days.
Then place tissues in the following solution: Formalin 10 c.c., pyridin 10 c.c.,
acetone 25 c.c., absolute alcohol 25 c.c., distilled water 30 c.c. Keep in this
solution for five days at room temperature. Then wash in water for one day.
Transfer to 95% alcohol for three days and then wash in water for one day. Put
tissue in dark bottle in i^% aqueous solution of silver nitrate for five days at room
temperature. Wash in distilled water for five to six hours. Transfer to reducing
mixture of 95 c.c. of 4% aqueous solution of pyrogallol and 5 c.c. of formalin. Keep
in this solution twenty-four hours. Wash in water and put through alcohol and
xylol. Imbed in paraffin.

Romanowsky. — Staining sections with Romanowsky stains is not very satis-
factory. The differential staining seems to fade out in passing through the alcohols.
This may be avoided by blotting the section after staining and differentiation and
then applying the xylol to the blotted section. After staining with Giemsa's stain
for ten to fifteen minutes, differentiate with i to 500 acetic acid. When the section
has a pinkish tinge, wash in water, dry, clear in xylol, and mount.

Good tissue staining may be gotten with Wright's stain. After removing the
paraffin with xylol and the xylol with absolute alcohol, pour on a sufficient number of
drops of stain and after one minute dilute with an equal number of drops of water.
Allow the diluted stain to remain for three to five minutes. Next wash in water,
differentiate, until the tissue has a pinkish tinge, in i to 500 acetic acid. This
differentiation is best done in a tumbler of the dilute acetic acid.

After washing in water, quickly pass through 95% and absolute alcohol, clear in
xylol, and mount.

I now use the panoptic method for staining tissues. In this I stain with Wright's
stain as given above but following the washing of the section we treat this with a
dilute Giemsa (1-15) for ten to fifteen minutes. Then wash and differentiate in i to
1000 acetic acid in water in a small beaker. When the section assumes a pinkish
tinge wash in tap water, then in 95% and absolute alcohol and clear in xylol.
Then mount in liquid petrolatum, immersion oil or balsam.

Skin Sectioning. — Of all tissues that of skin offers the greatest difficulty in pre-
paring sections. The best results can probably be obtained by fixation in picro-
sublimate (saturated aqueous solution picric acid i part; saturated aqueous solu-
tion bichloride of mercury one part); to this stock mixture add 5% glacial acetic
acid just before using. Fix small pieces of skin six to eighteen hours. Transfer
direct to 70% alcohol in which the tissue may be kept indefinitely.

For sectioning run through alcohols to absolute and then to a mixture of absolute
alcohol and carbon bisulphide (equal parts). Leave until tissue sinks, then transfer
to pure carbon bisulphide until tissue sinks. Then transfer to a saturated solu-
tion of paraffin in carbon bisulphide and thence to paraffin. Bisulphide of carbon

APPENDIX

449

has the disadvantage of foul odor and inflammability but does not seem to render
tissues brittle and difficult to section as does xylol.

NEUROLOGICAL STAINING METHODS

Neuropathology practically dates from the introduction of Marchi's method of
staining in 1885.

Ordinary osmic acid stains both normal and pathological fat. With Marchi's
method only the oleic acid of fatty degeneration is stained.

The method is not useful until three or four days have elapsed from the onset of the
condition causing the degeneration and it is applicable for only three or four months
because by that time phagocytes have taken up the pathological fat which is stained
in the Marchi method. The Weigert method is the one to use after a period of three
or four months. In Weigert's stain only the normal myelin sheath is stained and the
lack of staining of myelin sheaths in degenerated areas is the basis of the stain.
For demonstrating axonal reactions or other degenerative changes in nerve-cells, as
shown by bulging of the concave sides of the cells, eccentric nucleus and granular
appearance of the tigroid bodies, Nissl's method is the best.

For neuroglia fiber staining Mallory's phosphotungstic acid haematoxylin is to
be recommended.

I. For Marchi's Method.— Small pieces of nerve tissue are hardened in Miiller's
fluid for seven to ten days and are then transferred to a mixture of two parts Muller's
fluid and one part of a i% osmic acid solution and should remain in this mixture
for about seven days. The tissue thus treated is run through alcohols and imbedded
in paraffin in the usual way.

II. For Weigert-Pal Method. — Thin slices of tissue are fixed in 10% formalin
in about four days. The tissue should then be transferred to 5 % potassium bichro-
mate for about twelve days. The tissue is then imbedded and sections cut. If
only recently mordanted these sections may be at once stained with Weigert's
haematoxylin for twelve to twenty-four hours (10 c.c. ripened 10% solution haema-
toxylin in absolute alcohol and 90 c.c. water). Wash in water to which about 2%
of a saturated solution of lithium carbonate has been added. Now differentiate
from one-half to five minutes in %% solution of potassium permanganate until
the gray matter looks a brownish yellow. Next treat sections with oxalic acid
i gram, potassium sulphate i gram and water 200 c.c. until the gray matter is
almost colorless. This takes only a few seconds. Wash in water, pass through
alcohols and xylol and mount in balsam.

III. For Nissl staining either thionin or Giemsa staining is satisfactory.

IV. For neuroglia fiber staining use Mallory's Phosphotungstic acid haematoxylin.

Take of haematein ammonium, o . i gram.

Water, loo.oc.c.

Phosphotungstic acid crystals (Merck) 2 . o grams.

Dissolve the haematein in a little water with the aid of heat, and add it after it is
cool to the rest of the solution; no preservative is required. If the solution stains
weakly at first, it may be ripened by the addition of 5 c.c. of a Y±% aqueous
solution of potassium permanganate, or it may be allowed to stand for a few weeks
until it ripens spontaneously.
29

450 APPENDIX

MAKING AND STAINING OF FROZEN SECTIONS

The various types of ether freezing microtomes are not very satisfactory when
only used occasionally. With the general introduction of cylinders containing
compressed carbon dioxide, which is used for aerating waters, we have at hand a
practical and convenient method of making frozen sections.

The instrument makers furnish a freezing microtome of the Bardeen type whicl
can be attached directly to the cylinder by a revolving clamp nut.

It is necessary to have a stand to support the iron cylinder in a horizontal posi-
tion. The tissue, which may be taken at operation for immediate diagnosis, or
which preferably has been fixed in formalin for twelve to eighteen hours is immedi-
ately placed in water. If the tissues have been in alcohol it will require hours of
washing before they can be frozen. The piece of tissue which is to be frozen should
not be more than one-fifth of an inch thick. Having placed the piece of tissue on
the freezing box of the microtome we turn the valve* of the cylinder to allow the
gradual escape of gas. When frozen solid, we elevate the freezing box holding the
frozen tissue, by revolving a graduated disk with the left hand. In the right hand
we firmly grasp a well-sharpened blade of a carpenter's plane mounted in a wooden
handle. This is held at an angle of 45 degrees to the polished ways of the micro-
tome. By alternate shoving and withdrawing of the blade, held rigidly, we accumu-
late on the blade a number of sections. Then dip the blade in a vessel of water to
detach the sections which float in the water. Keep repeating the process until
numerous satisfactory sections are obtained. Handles for holding the Gillette
razor blades are good substitutes for the carpenter's plane.

These sections may be picked up with a strip of cigarette paper and applied to a
clean slide upon which a very small loopful of albumin fixative has been smeared
out with 30% alcohol. The piece of cigarette paper with the section underneath
is then firmly smoothed out upon the slide with filter-paper. The piece of cigarette
paper is then carefully stripped off and the section remains attached to the slide.
By careful heating over a very small flame, until the vapor just arises, the section
is fixed to the slide and we can then stain the section in any way that may be desired.

NOTE. — The procedures for carrying the tissues through celloidin are not given
as it requires perfect condition of the entire cutting surface of the microtome knife
and a considerable time for the passage through reagents and celloidin. It is more
suitable as a method when sections for class work are to be prepared.

B— MOUNTING AND PRESERVATION OF PATHOLOGICAL SPECIMENS
AND ANIMAL PARASITES

To Mount Small Round Worms. — Wash the hook, whip, or filarial worm in salt
solution, then drop in 70% alcohol containing 5% of glycerine; the glycerine-alcohol
mixture being at a temperature of 6o°C. When cool, pour into Petri dishes and
allow the alcohol to evaporate in the 37°C. incubator.

Mount in glycerine jelly, preferably in a concave slide, and ring the preparation
with gold size. The following is the formula for Kaiser's glycerine jelly: Soak one
part of gelatin in six parts of distilled water for two hours. Then add seven parts
of glycerine. To the mixture add i% of carbolic acid, warm for fifteen minutes,
with constant stirring, and then filter through cotton.

APPENDIX

451

To Prepare Tape-worms. — Wash in salt solution. Wrap around a piece of glass
as a glass slide and fix in salt solution containing 2 to 5% of formalin. Then keep
the preparation permanently in 70% alcohol. If preferred, the specimen may be
run through alcohols and xylol and mounted in balsam.

Larvae. — Mosquito larvae may either be prepared as for small round worms or
they may be dropped into 70% alcohol at 6o°C. and then passed through alcohols
and cleared in xylol and mounted in balsam. Flukes and insects may require treat-
ment with hot (60° to 7o°C.) solution of 10 to 20% sodium hydrate solution. Then
wash thoroughly in water and subsequently pass through alcohols to xylol and mount
in balsam. Clove oil or cedar oil clears more slowly, but makes specimens less brit-
tle than does xylol. Another satisfactory method is to drop insects or larvae into
acetone at 6o°C. and after being in this from one to twelve hours to clear in xylol
or clove oil and mount in balsam.

Nematodes. — Looss has a method of first washing a small nematode or delicate
fluke in salt solution. Then pouring this first salt solution out of the test-tube in
which the washing was carried out, to add fresh salt solution, and then an equal
amount of saturated aqueous solution of bichloride of mercury. The shaking is
easily carried on in the test-tube. After washing in water the worm is passed
through alcohols, one strength of which should contain iodine. Clear in xylol and
mount in balsam.

An excellent method is that of Langeron.

After washing in salt solution fix for a few hours in 5% formalin. Then transfer
to lactophenol which has been diluted with an equal amount of water. Allow to
remain in this solution for several hours and then transfer to pure lactophenol in
which fluid the specimens are to be mounted. Ring with paraffin or with gold size.
(To make lactophenol take 2 parts of glycerine and i part each of distilled water,
crystallized carbolic acid and lactic acid.)

A quick method of preparing small nematodes for examination is to fix them for
from two to twelve hours in 5 to 10% formalin, this being heated at 6o°C. at the
time the worms are dropped into it. Then transfer to the following solution:

Glucose syrup (glucose, 48; water, 52), 100 c.c.

Methyl alcohol, 20 c.c.

Glycerine, 10 c.c.
Camphor, q.s. (a small lump for preservation).

They may be mounted directly in this and the cover-slip ringed with about 6o°C,
paraffin, followed with gold size.

Preparations so cleared and mounted in glycerine jelly should also be ringed with
paraffin or some cement.

Flukes, cestodes, and nematodes are best stained with carmine. The following
is a good formula.

Dissolve, by boiling, 4 grams carmine in 30 drops HC1 and 15 c.c. water. Then
add 95 c.c. of 85% alcohol and filter while hot. Neutralize with ammonia until
precipitate begins to form. Then filter cold.

i. Stain parasites taken from 70% alcohol for five to 'twenty minutes. 2. Dif-
ferentiate in 3% hydrochloric acid. 3. Pass through alcohols to xylol and mount in
balsam.

452

APPENDIX

Mites, Fleas and Various Small Insects. — By simply taking i or 2 drops of
liquid petrolatum and mounting the specimen in it then covering with a cover-
glass one is able to study the details of these objects almost as well as if they were
passed through acetone and xylol into balsam. Liquid petrolatum is also most
excellent for mounting the aerial hyphae of fungi with their sporangia as well as for
Romanowsky stained blood smears.

Pathological Specimens. — Pathological tissues which are to be sent to a labora-
tory for sectioning or to be kept for future study should be fixed by one of the
methods given in Section A of the appendix.

Formalin fixation is the more convenient — that with Zenker's fluid the more
perfect. After fixation with Zenker's fluid the pieces of tissue must be washed in
running water over night.

After fixation the pieces of tissue are transferred to 70% alcohol in which they ma)
be kept indefinitely.

For preservation of gross specimens the method of KAISERLING is generally use<

Fix for from one to five days in Solution i.

Solution I

Formaldehyde, 200 c.c.

Water, 1000 c.c.

Nitrate of potassium, 15 grams.

Acetate of potassium, 30 grams.

The position of the specimen should be changed from day to day. There must
be at least five times as much fluid as specimen. Drain and transfer to 80% alcohol
for a few hours, then into 95% alcohol until the natural color is just restored.

Finally preserve in

Acetate of potassium, 200 grams.

Glycerine, 400 c.c.

Water, 2000 c.c.

It is advisable to keep these specimens in the dark as light destroys the natui
color.

To Prepare Flies or Mosquitoes for Transmission through the Mails.— Wrap
the insect carefully in a piece of tissue paper (toilet-paper answers). Impregnate
sawdust with 5% carbolic acid solution and fill around the tissue paper in the box
containing them. (Barely moisten.)

It is very satisfactory to take a tube form vial with a cork from the inner sur-
face of which two small shallow holes have been bored, one containing parafor-
maldehyd, the other camphor. The insect is mounted upon a pin stuck in the
cork, which latter is inserted and paraffined externally.

C— PREPARATION OF NORMAL SOLUTIONS

A normal solution is one which contains the hydrogen equivalent of an element,
expressed in grams, dissolved in sufficient distilled water to make 1000 c.c.
hydrogen equivalent is the atomic weight of any element divided by its valence. Ii
a base, salt or acid, we use the molecular weight in grams divided by valence.

APPENDIX 453

What may be considered as the valence of a base is shown by the number of
hydroxyls combined with it; that of an acid by the number of replaceable hydrogen
atoms which it contains.

To make a normal solution, dissolve in distilled water a weight in grams equal
to the sum of the atomic weights of the substance divided by its valence, and make
up the volume to exactly 1000 c.c.

NaOH is univalent. Na = 23. O = i6. H=i. Dissolve 40 grams NaOH in
water and make up to exactly 1000 c.c.

Oxalic acid is COOH— COOH+2H2O which gives it a molecular weight of 126.
As it contains two carboxyl groups it is dibasic, and it is necessary to divide the
molecular weight by 2, so that for a normal solution of oxalic acid we dissolve 63
grams in a volume of distilled water made up to 1000 c.c.

If a chemical laboratory is not accessible one may prepare normal solutions with
an error so slight as to be unimportant in clinical work in the following way:

Sodium hydrate being very hygroscopic, it is impossible to accurately prepare a
normal solution by directly weighing out the substance. Instead, select perfect
crystals of oxalic acid, such as can be obtained in a drug store, and weigh out on the
most accurate apothecary scales obtainable exactly 6.3 grams of the most perfect
crystals in the bottle. Put these preferably in a volumetric flask and make up with
distilled water to 1000 c.c. Less accurate is the use of a measuring cylinder. If
care is used this should give N/io solution of oxalic acid in which the error is less
than i%.

Having N/io acid at hand, we may prepare N/io NaOH in the following way:
Weigh out an excess of sodium hydrate (5 grams of stick caustic soda) and dissolve
in 1 100 c.c. of distilled water. Take up 10 c.c. of this solution with a pipette and let
it run into a beaker. Add 6 drops of phenolphthalein solution. This gives a violet-
pink color. Fill the burette with the N/io oxalic acid solution and let it run into
the sodium hydrate solution in the beaker until the pink is just discharged. Reading
off the number of cubic centimeters of the N/io acid used, we know the strength
of the sodium hydrate solution. It is well to repeat the titration and take an
average.

If 10.5 c.c. of the oxalic acid solution were required it would show that the sodium
hydrate solution was stronger than N/io, as only 10 c.c. would have been necessary
if the NaOH solution had been N/io. It is therefore necessary to dilute the sodium-
hydrate solution in the proportion of 10 to 10.5. Measure exactly 1000 c.c. of the
too concentrated sodium-hydrate solution and add to it 50 c.c. of distilled water, mix
thoroughly, and we have 1050 c.c. of N/io solution of NaOH. icooX 10.5 = 10,500.
1 0,500 -1-10 = 1050.

As Acidum hydrochloricum U. S. P. is about two-thirds water (68.1%) to make
N/io HC1, which would require 3.65 in 1000 c.c., it would be necessary to take about
three times this amount of U. S. P.^cid. Take 12 c.c. of the acid and add distilled
water to make 1 100 c.c. Put 10 c.c. of this dilute solution in a beaker. Add phenol-
phthalein solution and titrate. If n c.c. of N/io NaOH were required it would be
necessary to add 100 c.c. of water to a volume of 1000 c.c. of the diluted hydrochloric
acid. ioooXii = n,ooo-T-io = iioo.

Other acid and alkali solutions can be made as for N/io HC1 and N/io NaOH.

454 APPENDIX

D— CHEMICAL EXAMINATION OF THE BLOOD

Blood Sugar. — Normally we have about 0.1% sugar, or 100 mg. in 100 c.c. of
blood. In diabetes we have hyperglycaemia with an increase of blood glucose
to twice or even eight times as much. The determination of blood sugar is impor-
tant in differentiating the so-called "renal diabetes" from true diabetes. In renal
diabetes we have a normal blood sugar content. Furthermore, the sugar in the urine
rarely exceeds i % and this glycosuria is not affected by variations in carbohydrate
intake as is the case with true diabetes mellitus. Furthermore, there are no symp-
toms, such as thirst, excessive polyuria, loss of weight, etc.

The micro-method of Bang is the one used by many workers in making this deter-
mination. Objections to it that we have noted have been the necessity for an accu-
rate chemical balance for the weighing of the drop or two of blood which is taken
up on a previously weighed piece of filter-paper. Again the titration of the cuprous
chloride with N/2oo iodine solution has to be carried out with exclusion of air and
furthermore, the previous boiling gives changing results according to time of process.
In my opinion it can only be carried out accurately in the hands of an experienced
chemical worker.

Myers and Fine Modification of Lewis and Benedict Method. — For taking blood,

whether for sugar or nonprotein nitrogen determination, we use the blood system

"described under "Blood Examination." With a graduated centrifuge tube we.

make a blue pencil mark at 2 c.c. for sugar determinations, or at 5 c.c. for nonprotein

nitrogen ones.

A small pinch of finely powdered potassium oxalate is dusted into the bottom
of the graduated centrifuge tube. As the blood drops into the tube we keep agitating
the tube so that the oxalate dissolves and prevents coagulation of the blood. In-
stead of the powdered potassium oxalate we may put i c.c. of a 2% solution of the
oxalate in the centrifuge tube and make a mark for the blood at the 3 c.c. or 6 c.c.
line. Folin uses a 2 or 5 c.c. pipette into which potassium oxalate has been dusted
and which is connected with the needle in the vein.

Transfer the 2 c.c. of oxalated blood, to which has been added exactly 8 c.c. of
water, to a test-tube. Then add 0.2 gram of picric acid. Mix thoroughly and after
standing five minutes filter through small dry filter. Three c.c. of filtrate are placed
in a tall test-tube and i c.c. of 20% Na2CO3 added. Tube is placed in boiling water
bath for fifteen to thirty minutes. Now cool and make volume up to 20 c.c. and
compare by Duboscq colorimeter with a known dextrose solution (0.1%) similarly
treated.

Instead of the Duboscq or Hellige colorimeter one can use the following method
which is sufficiently accurate for clinical purposes.

In a case of diabetes make up a 0.2% sugar solution instead of the 0.1% and treat
as above with the picric acid. Then make up accurately to 20 c.c. in a graduated
cylinder. Now with the 20 c.c. of yellow-colored solution from the blood in a
tube between the thumb and forefinger of the left hand we add to the empty
tube alongside the known 0.2% solution from the graduated cylinder. When the
colors match we read off the amount used from tfce cylinder, and calculate the
strength of the blood sugar tube. If i$ c.c. were required it would show that the
sugar of the blood equalled 0.15%.

APPENDIX

455

Nonprotein Nitrogen of Blood. — Normally we have from 25 to 35 mg. of non-
protein N in 100 c.c. of blood. This is greatly increased in chronic nephritis and
especially in uraemia. The nonprotein N is more easily and accurately determined
than the urea N. Of course, the urea N makes up about one-half of the normal
nonprotein N and in uraemic conditions it may constitute from 80 to 90% of the
nonprotein N. Hawk gives urea N as 12 to 23 mg. per 100 c.c. of blood. In
uraemia it may amount to 70 to 300 mg. with a nonprotein N increase of from 90
to 350 mg. per 100 c.c. of blood.

To Estimate Nonprotein N. — Take 5 c.c. blood in a graduated centrifuge tube
with powdered potassium oxalate as for blood sugar. Be sure not to use ammonium
oxalate. Now gradually add, with constant stirring, 75 c.c. of absolute alcohol,
mix thoroughly, filter through dry filter, wash precipitate with several small portions
of alcohol, evaporate filtrate and washings to dryness on water-bath, using a small
porcelain dish for the purpose, and with smallest possible quantity of water take up
and transfer residue to a small flask. Add i to 2 grams potassium sulphate and 2
c.c. of concentrated sulphuric acid, place over a small flame and heat until all water
is driven off and the contents of flask, after having become thoroughly charred,
have again become a pale yellow or colorless. Cool, add about 20 c.c. of water,
again cool, add 4 or 5 drops of phenolphthalein, gradually add 10% solution of sod-
ium hydrate, keeping contents of flask cool by frequent immersion in cold water,
until exact point of neutrality is reached. Now add 5 grams of neutral potassium
oxalate, stir until dissolved and then add about 10 c.c. of neutral 20% formaldehyde
(50% formalin) and then from burette add N/50 NaOH until permanent pink
color is obtained.

The number of cubic centimeters N/50 NaOH required times 0.28 equals number
milligrams nonprotein nitrogen in 5 c.c. of blood.

Urea N of Blood. — The following method suggested by Dunning has been
modified in our laboratory. For urea estimation take 5 c.c. blood as for nonprotein
N and dilute with 25 c.c. distilled water and add 30 grams of crystallized sodium
sulphate or 13 grams of anhydrous sodium sulphate. The mixture is then warmed
to 35°C. when the sodium sulphate is dissolved and the blood proteins precipitated.
The mixture is then made up to 50 c.c. with a saturated solution of sodium sulphate,
thoroughly shaken and allowed to stand a few minutes. It is then filtered and
25 c.c. of the filtrate used for the test and 10 c.c. for a control. Each ic.c.c. repre-
sents i c.c. of the oxalated blood.

To 25 c.c. of the nitrate add distilled water to 100 c.c. and one finely powdered
Urease-Dunning tablet and digest for i hour at 50° C. or over night at room tem-
perature.

After digestion filter through double dry filter and to 40 c.c. of this nitrate, which
represents i c.c. of the blood, add 2 drops of methyl orange.

Now add 2 drops of methyl orange to the control and titrate with N/2O HC1
to a faint pink color. Then titrate the urease-treated solution to exactly the same
shade of pink as the control.

The amount of N/20 HC1 required to neutralize the urease-treated solution, less
the amount required for the control, multiplied by 0.0015 will give the urea in i c.c.
of the blood: or multiplied by 0.0007 will give the urea nitrogen in the i c.c. of blood.

The end reaction is not at all sharp and requires considerable experience to deter-

456 APPENDIX

mine it. The control should be titrated before the test solution acted upon by the
urease and the shade of the test solution should correspond to the control. About

2 drops of a 0.05% aqueous solution of methyl orange should be used for 50 c.c. of
test solution.

Hydrogen-ion Concentration of the Blood. — Levy, Rowntree and Marriott have
proposed this determination as an index of the reaction of the blood. The test
may be made either on oxalated blood or with serum. The hydrogen-ion concen-
tration of the serum of normal persons varies from p H 7.6 to p H 7.8, that of oxa-
lated blood from p H 7.4 to p H 7.6. The exact point of neutrality, p H 7, is only
reached in severe uncompensated acidosis and a reaction of p H 8 is only possibly
obtainable after administration of alkalis.

One to 3 c.c. of clear serum or of blood is run, by means of a blunt-pointed
pipette, into a dialyzing sac which has been washed inside and outside with salt
solution and which has been tested for leaks by filling with the salt solution. The
sac is lowered into a small test-tube (100 x 10 mm., inside measurements) containing

3 c.c. of the salt solution, until the fluid on the outside of the sac is as high as on the
inside. From five to ten minutes are allowed for dialysis. The collodion sac is
removed and five drops of the indicator (an aqueous 0.01% solution of phenol-
sulphonphthalin) are thoroughly mixed with the dialysate. The tube is then com-
pared with the series of standards until the corresponding color is found, which
indicates the hydrogen-ion concentration present in the dialysate.

These tests may be carried out with 3 c.c. of blood or serum. The same results
are obtained with i c.c. of blood or serum on the inside of the sac, and with this
amount it is immaterial whether there is i or 3 c.c. of salt solution on the outside.

For the comparison of tubes with standards a good light (natural or artificial)
and a white background are requisites. Readings must be made immediately.
The tube matching most closely is selected and also the tubes on either side of it.
These are critically inspected against a white background. Changing the order
of the tubes often makes differences more apparent.

Hynson, Westcott and Co. prepare a set of standards with the h drogen-ion con-
centration marked on each ampule. The dialyzing collodion sacs are made as for
the Abderhalden test or they may be purchased.

E— CHEMICAL EXAMINATION OF URINE

For the prevention of decomposition when a urine is not examined shortly after
voiding, chloroform (10 to 20 drops added to a tightly corked bottle) or formalin
(4 or 5 drops to a pint of urine) are ordinarily employed. Formalin is better for
microscopical material, but, owing to its reducing power, should be substituted by
boric acid in urine to be examined for sugar. For clearing urine, turbid by reason
of bacteria, rubbing up with Talcum purificat., U. S. P., and filtering is recom-
mended. // is advisable to call for a fresh specimen and not to examine a decomposed
urine either for casts or albumin,

A twenty-four-hour specimen is necessary for accurate work. The urine should
be collected in clean separate bottles. Where pus comes from the bladder the
proportion of pus in each bottle will be practically the same; if from the kidneys
the amount will vary in the different bottles.

ter

APPENDIX 457

The amount of urine varies in different individuals (water or beer habit). It is
usually given as from 1000 to 1500 c.c.

Long proposes to substitute 2.6 for Haeser's coefficient which, if multiplied by
the two final figures of the specific gravity taken at 25°C., gives the weight of urin-
ary solids in 1000 c.c.

Albumin. — Practically serum albumin alone is clinically important.

The two usual tests are i. Heat test and 2. Heller's nitric acid test. For the
former, add 3 to 10 drops of 5% acetic acid to the perfectly clear urine in a test-
tube and bring to a boil. By boiling the upper portion a turbidity in contrast with
the clear lower portion may be obtained. I usually prefer to heat the urine and after
the boiling add the 5% acetic acid, drop by drop. This will clear up the turbidity
due to phosphates.

A more delicate test for albumin is the following: Add to a test-tube half filled
with filtered urine one-fifth its volume of a saturated aqueous solution of sodium
chloride; heat to the boiling-point; add 2 to 5 drops of 50% acetic acid and
heat again. This test may serve to distinguish nucleo-albumin, as most forms of
nucleo-proteid found in urine do not react to the test, while serum albumin does.
Thus where a positive nitric acid test is present, and no precipitate occurs with
this test, the proteid present is usually nucleo-proteid.

For Heller's test, pour a small amount of nitric acid into a narrow test-tube
and, while holding the tube at an angle of about 45 degrees, superimpose a layer of
the urine to be tested, which is delivered drop by drop from a pipette and allowed
to flow down the side of the tube.

This test can be converted into a quantitative one which is sufficiently accurate
for clinical purposes. It is based on the fact that a specimen of urine containing
0.003 % of albumin will give a perceptible ring at the layering of the urine and acid
in two minutes. If the ring appears at once or in a few seconds" the albumin con-
tent is greater. From the qualitative test an idea can be formed as to the amount
of albumin which the urine contains, a heavy ring forming immediately showing a
considerable albumin content. Probably the highest elimination of albumin is
found in chronic parenchymatous nephritis where it may run from i to 3%. In
an ordinary case of acute nephritis 0.5% would be an average content.

Recently I have been using for both qualitative and quantitative albumin tests
the apparatus shown in Fig. 7. This is simply a 5-inch piece of ^-inch soft
glass tubing heated at a point 2 inches from one end, drawn out about 2 inches
and bent to form a U tube with one end shorter than the other. This form of
tube enables one to perform two tests with the same column of nitric acid and is
easily cleaned and dried. They may be kept suspended around a glass tumbler's
rim. Taking up a small amount of nitric acid with a capillary bulb pipette it is
deposited in the capillary curve of the bent tube. This acid pipette should be
kept attached to the acid bottle. With a second pipette the urine is deposited in
the short arm of the U tube and the presence of albumin shows by a distinct ring
at the junction of urine and acid in the clear capillary tubing. The long arm will
serve for the introduction of a different specimen of urine for the albumin test.

For quantitative test we dilute the filtered urine with one or more parts of nor-
mal salt solution according to the intensity of the albumin ring. A very convenient
way of making the dilution is with a graduated centrifuge tube. Make a i to

458 APPENDIX

t in the

10 dilution of the urine, mix and draw up with a bulb pipette and deposit i
short arm of the U tube containing nitric acid. A distinct ring forms in two to
three seconds. Pour off one-half of the diluted urine and make up with an equal
amount of saline. Deposit this i to 20 dilution in the long arm. The ring forms
in about a minute. With further testing it is found that a i to 40 dilution shows a
perceptible ring in just two minutes. This final and successful dilution multiplied
by 0.0033 gives the percentage of albumin in the urine (40 X 0.0033 = 0.13%).

Should it be desired to determine the nature of the proteids present either in
urine or in exudates or transudates the following method is applicable. Determine
the percentage of total proteid by the method employed above. Then throw down
the globulins by the addition of an equal amount of a saturated solution of ammo-
nium sulphate, filter and estimate the proteid content of the filtrate. The differ-
ence between that and the total gives the percentage of globulin. The filtrate is
now treated with 5% acetic acid until a precipitate of nucleo-proteid ceases to form;
the fluid is filtered and the clear filtrate (which should not show any turbidity with
a drop of 5% acetic acid) is tested for its proteid content, which represents the
serum albumin. When the combined percentage of globulins and serum albumin
is subtracted from the total proteid percentage we have the percentage of nucleo-
proteid.

Esbach's Quantitative Method for Albumin. — The use of the Esbach tube is at-
tended with some uncertainty, whether using the original Esbach solution or that
devised by Tsuchiya, the precipitate at times refusing to settle. The method of
using the tube is to add the urine to the U mark and then the reagent to the R mark,
mix, and allow to stand in upright position. If Esbach's reagent has been used the
reading is made at the end of twenty-four hours, but when Tsuchiya's reagent is
employed the reading is made at the end of two hours. The number at the end of
the line which corresponds to the upper limit of precipitate will be the number
of grams of dry albumin per liter of urine. Esbach's reagent consists of 10 grams
of picric and 20 grams of citric acid dissolved in i liter of water. Tsuchiyas'
reagent is made by dissolving 1.5 grams of phosphotungstic acid in a mixture of 5
c.c. strong hydrochloric acid and 95 c.c. of 95% alcohol. In this method also
dilution must be resorted to if albumin or specific gravity are excessive.

Nucleo -protein. — Increased quantities of this protein occur in pyelitis, nephritis,
and inflammations of the bladder.

For its detection albumin, if present in any considerable quantity, must be re-
moved by boiling as described above. Then place 10 c.c. of this urine in a small
beaker, dilute with 3 volumes of water and make the reaction very strongly
acid with acetic acid. If the solution becomes turbid it is an indication that
nucleo-protein is present.

Bence-Jones Body. — (Albumose.) Perform the heat test for albumin. The
appearance of a heavy precipitate which partially clears on boiling suggests albu-
mose. If albumose is present a cloud will appear in the filtrate on cooling. The
precipitate formed with nitric acid, if due to albumose, disappears with heat, that
of serum albumin does not.

As another test for the Bence-Jones body, usually present in multiple myelomata,
that of Boston is of value. Mix 15 c.c. urine in a test-tube with an equal amount of
saturated NaCl solution. Add 2 c.c. of 40% NaOH solution and shake the contents

APPENDIX 459

of the tube thoroughly. Heat the upper contents of the tube to boiling and add lead
acetate solution (10%) drop by drop continuing the heating.. A brown to black
precipitate (sulphur) shows this form of albumin.

In tests requiring the removal of albumin boil 'the urine and add dilute acetic
acid until the precipitate is flocculent, then filter.

SUGAR

Fehling. — Pour equal parts of Fehling's copper solution (34.639 grams of copper
sulphate in 500 c.c. of water) and Fehling's alkali solution (173 grams sodium potas-
sium tartrate and 50 grams sodium hydrate in 500 c.c. water) into a test-tube. Mix
and dilute the deep blue solution with 2 parts of water. Heat the upper portion
of the diluted Fehling's solution in the flame to boiling and drop in from a pipette
the urine to be examined. A yellowish to red precipitate shows the presence of
sugar.

Fehling's test will show the presence of J^oo of i% of glucose in an aqueous
solution but is vastly less delicate for sugar in urine. This is due to the power
of the creatinin in urine of holding the reduced suboxide of copper in solution.
An important point is that the creatinin is broken up by prolonged boiling hence
the puzzling precipitates one gets at times after a long period of boiling are explained
in this way. Glycuronic acid may cause a doubtful reaction. If the precipitated
cuprous oxide is in very fine granules the color is greenish, if less fine, greenish
yellow and if quite coarse, reddish.

Creatinin holds in solution the copper suboxide formed by uric acid as well as
that resulting from very small glucose content of urine.

As a test for doubtful glycosuria it is well to give 100 grams of pure glucose. A
normal person should deal with such an amount without showing sugar reaction of
the urine.

Phenylhydrazin (Kowarsky). — Mix 5 drops of pure phenylhydrazin in a test-
tube with 10 drops of glacial acetic acid. Shake lightly and add 15 drops of satu-
rated solution of NaCl. This makes a pasty mixture. Now add 10 c.c. of the urine
and bring carefully to a boil over a small flame and continue to boil gently for two
minutes. Upon cooling a yellowish crystalline precipitate falls more or less rapidly
according to the sugar content of the urine. If the urine contains 0.2% or more of
sugar the precipitate appears in a few minutes. The test is sensitive for 0.03% of
sugar.

Fermentation Test.— This is the surest test for sugar in the urine. It will show
the presence of 0.05% of glucose. Instead of the Einhorn apparatus one may be
extemporized by taking a 50 c.c. cylinder, filling it to overflowing with the urine
which has previously been rubbed up with a piece of compressed yeast the size of a
hazel nut. The urine-should be made acid with tartaric acid to prevent ammoniacal
decomposition with the formation of CO2. A small 3-inch test-tube is filled with the
yeast-treated urine and dropped mouth downward into the 50 c.c. cylinder. The
apparatus is incubated for twenty-four hours and the presence of gas in the closed
end of the test-tube shows that sugar was present. A control to determine that the
yeast does not contain sugar is advisable. To utilize this test as a quantitive one,
first accurately take the specific gravity of the urine; then add the yeast and fill

460 APPENDIX

the test-tube and cylinder as directed above. Next pour off or pipette
urine exactly to the 50 c.c. mark. Incubate for twenty-four to forty-eight hours
and make up the loss by evaporation, with distilled water. After the urine has
cooled down to room temperature the contents of tube and cylinder are thoroughly
mixed (the small tube having been withdrawn with a pair of forceps), then filtered
to remove the sediment of yeast and then brought to the exact original volume of
50 c.c. with distilled water to make up the loss by evaporation. (If there should be
doubt as to the completion of the fermentation of the glucose a qualitative test for
sugar can be made.) The specific gravity is again taken and the difference between
this and the first reading multiplied by 0.23. Example: Specific gravity of unfer-
mented urine, 1.030, that of urine after incubation, 1.022. Difference, 8X0.23 =
1.84%.

It is advisable to have two good urino meters, one to register from 1000 to 1025,
a second to register from 1025 to 1050.

Benedict's New Method for Quantitative Determination of Sugar in Urine.

The solution for quantitative work has the following composition: .

Copper sulphate (pure crystallized) 18 .o gm.

Sodium carbonate — crystallized (100 grams of anhydrous salt

will answer) 200 . o gm.

Sodium or potassium citrate 200.0 gm.

Potassium sulphocyanate 125 .o gm.

5% potassium ferrocyanid solution 5.0 c.c.

Distilled water to make total volume of 1000 . o c.c.

With the aid of heat dissolve the carbonate, citrate and sulphocyanate in enough
water to make about 800 c.c. of the mixture, and filter if necessary. Dissolve the
copper sulphate separately in about 100 c.c. of water and pour the solution slowly
into the other liquid, with constant stirring. Add the ferrocyanid solution, cool and
dilute to exactly i liter. Of the various constituents, the copper salt only need be
weighed with exactness. Twenty-five c.c. of the reagent are reduced by 50 mg. of
glucose.

Sugar estimations are conducted as follows: The urine, 10 c.c. of which should
be diluted with water to 100 c.c. (unless the sugar content is believed to be low), is
poured into a 50 c.c. burette up to the zero mark. Twenty-five c.c. of the reagent
are measured with a pipette into a porcelain evaporating dish (25-30 cm. in diameter),
10 to 20 grams of crystallized sodium carbonate (or one-half the weight of the anhy-
drous salt) are added, together with a small quantity of powdered pumice-stone or
talcum, and the mixture heated to boiling over a free flame until the carbonate has
entirely dissolved. The diluted urine is now run in from the burette, rather rapidly
until a chalk white precipitate forms, and the blue color of the mixture begins to
lessen perceptibly, after which the solution from the burette must be run in a few
drops at a time, until the disappearance of the last trace of blue color, which marks
the end point. The solution must be kept vigorously boiling throughout the entire
titration. If the mixture becomes too concentrated during the process, water may
be added from time to time to replace the volume lost by evaporation. The cal-

APPENDIX 461

culation of the percentage of sugar in the original sample of urine is very simple.
The 25 c.c. of copper solution are reduced by exactly 50 mg. of glucose. Therefore
the volume run out of the burette to effect the reduction contained 50 mg. of the
sugar. When the urine is diluted i :io, as in the usual titration of diabetic urines,
the formula for calculating the per cent, of sugar is the following:
0.050
•.£-- times 1000 equals per cent, in original sample,wherein X is the number of

cubic centimeters of the diluted urine required to reduce 25 c.c. of the copper
solution.

In the use of this method chloroform must not be present during the titration.
If used as a preservative in the urine it may be removed by boiling a sample for a
few minutes, and then diluting to its original volume.

This solution will keep indefinitely and it is claimed by Benedict, that compari-
son with the polariscope and by Allihn's gravimetric process will show it to be more
accurate than any of the ordinarily used methods.

APPROXIMATE QUANTITATIVE ESTIMATION with Fehling's solution. (One c.c. of
Fehling's solution is reduced by 5 mg. glucose.)

Measure off 2 c.c. of Fehling's solution in a pipette and put in a test-tube or
small beaker and dilute with 20 c.c. of water.

Bring the diluted Fehling's to boiling and drop in drop by drop the urine from
a dropping-bottle for which the number of drops per cubic centimeter has been
noted. Estimating 20 drops to the cubic centimeter if 2 drops of urine are re-
quired to reduce the copper it would show a sugar percentage of the urine of 10.
Four drops 5%, 8 drops 2.5%, 16 drops 1.25%, 32 drops 0.6%, 64 drops 0.3%, 100
drops 0.2%.

Nylander's Bismuth Reduction Test. — Put 5 c.c. of urine in a test tube and add
0.5 c.c. Nylander's reagent, then heat for five minutes in a boiling water bath. If
sugar be present the mixture will darken to become black on standing. For the
reagent digest 2 grams of bismuth subnitrate and 4 grams Rochelle salts in 100 c.c.
of 10% KOH solution. Cool and filter.

Urinary Tests in Connection with Acidosis

The determination of the ammonia quotient, which is the ratio of N eliminated
as ammonia to total nitrogen elimination, has assumed great importance by reason
of its connection with various forms of acid intoxication, as in diabetes, pernicious
vomiting of pregnancy, and various hepatic diseases.

The degree of acidosis is better determined by the quantitative estimation of
nitrogen elimination as ammonia than by estimating quantitatively the amount of
diacetic and /3-oxybutric acid in the urine. Normally we have about 0.7 gram of
ammonia eliminated daily. In acidosis this may rise to 5 or 10 grams and instead
of being from 3 to 5% of the total N, it may amount to 30 to 50%.

In the acidosis connected with chronic nephritis it has been found that the pre-
formed ammonia is often below normal, indicating a defect in the normal neutraliz-
ing action of ammonia. Where there is excessive formation of acid bodies as in
diabetes, or when liver cell degeneration interferes with the normal conversion of
ammonia into urea we have an increase in urinary ammonia output.

462 APPENDIX

Reaction. — Urine may be quite acid in acidosis. The reaction of the mixed
twenty-four-hour excretions is acid to litmus. At times during this period, especi-
ally after meals, the reaction may be alkaline. It is not positively known to
what the acidity is due, some authorities considering it due to sodium dihydro-
gen phosphate, while others attribute it to organic acids. The acidity being very
largely due to food, naturally it would vary in health, as the nature of food varies.
The degree of acidity under normal conditions is such that from 40 to 60 c.c. of
N/i alkali will be required to neutralize the twenty-four-hour excretion.

The acidity can be determined by measuring 25 c.c. of sample into beaker, adding
75 c.c. of distilled water, and also, if available, 10 grams of neutral potassium oxalate
and finally 3 or 4 drops of phenolphthalein From a burette add N/io sodium
hydrate until a permanent faint red or pink color is obtained. High-colored urines
must be diluted with more than 75 c.c. of water before the end reaction can
readily be detected. Normally, 6 to 8 c.c. neutralizes 25 c.c. urine.

Formalin Method for the Estimation of Ammonia

Free ammonia reacts with formalin to form hexamethylenetetramine. If sodium
hydrate is added to neutralized urine in the presence of formalin free ammonia
is liberated and reacts with the formalin. So soon as all the ammonia has been
liberated, the end reaction occurs.

Ronchese first utilized this principle and Mathison found that potassium oxalate
made the end reaction sharper. Brown found that preliminary clearing with
lead subacetate made the end reaction still sharper and removed certain nitrogenous
substances which reacted with formalin making the result only about 5% higher
than with Schaffer's method. The technic is as follows: About 60 c.c. of filtered
urine are treated with 3 grams of basic lead acetate, well stirred, allowed to stand
a few minutes and filtered. The filtrate is treated with 2 grams of neutral potas-
sium oxalate well stirred and filtered; 10 c.c. of the clear filtrate are diluted to 50
c.c. with distilled water; a few drops of i% phenolphthalein solution are added.
The mixture will be slightly alkaline or acid. Five grams potassium oxalate
are added and stirred. It is exactly neutralized with decinormal NaOH or H2SO4.
Twenty c.c. of 20% conmercial formalin, previously made neutral, are added, and
the solution again titrated with decinormal NaOH to neutralization. Every cubic
centimeter of decinormal NaOH corresponds to 0.0017 gram NH8. The quantity
of ammonia is then calculated on the basis of the twenty-four-hour volume.
Example: The 10 c.c. of urine required 4 c.c. N/io NaOH to give a pink color.
4X0.0017 = 0.0068. Then 100 c.c. urine would contain 0.068 and 1000 c.c.
(twenty-four-hour urine amount) 0.68 gram of ammonia.

ESTIMATION OF TOTAL NITROGEN

Principle. — The nitrogenous material of the urine is converted into ammonium
sulphate on boiling with H2SO<. The ammonia is then estimated as described
under estimation of ammonia by the formalin method.

Technic. — Solutions required:

i. Twenty per cent, commercial formalin previously made neutral with NaOH.

APPENDIX

463

2. N/io NaOH.

3. Forty per cent. NaOH.

Ten c.c. of filtered urine are pipetted into a Kjeldahl or Koch flask; 10 c.c. of
concentrated H2SO4 and 10 grams K2SO4 are added. The mixture is heated over
a free flame, gently at first to avoid foaming, and is finally brought to a boil, which
is continued until the mixture is perfectly clear, usually requiring forty-five minutes
to an hour. The contents are cooled and quantitatively transferred to a 200 c.c.
volumetric flask and i c.c. of phenolphthalein
solution added. The greater part of the
acidity is now neutralized by adding about
30 c.c. of the 40% NaOH. It is cooled
under a water tap and made up to the 200 c.c.
mark; 10 c.c. are taken, diluted in 50 c.c. with
distilled water and exactly neutralized with
N/io NaOH. Twenty c.c. of the formalin
solution are now added and the titration
again performed. The pink end reaction is
beautifully clear and sharp. The second
reading multiplied by the factor 0.0014 gives
the amount of nitrogen in grams in 10 c.c.
of the fluid. It is tthen computed for the
twenty-four-hour volume as for N, eliminated
as ammonia. Example: It required 5 c.c.
N/io NaOH — 5X0.0014 = 0.007. As origi-
nal 10 c.c. were diluted to 200, the 10 c.c.
taken for titration would onlybe^o; hence
0.007X20 = 0.14 gram for 10 c.c. or 1.4 for
100 c.c. or. 14 grams for 1000 c.c.

UREA ESTIMATIONS

The amount of urea, which represents from
85 to 90% of the total nitrogen, is usually
determined instead of the total N. The
hypobromite and hypochlorite methods are,
however, lacking in accuracy, and more exact
methods of urea estimation are more time-con-
suming than the one just given for total N.

Probably the most convenient test for urea is the hypobromite method, using
the Doremus ureometer with a side tube connected to the closed arm of the fermenta-
tion tube by a glass stop cock.

The reagent is prepared by taking 70 c.c. of a 30% stock solution of NaOH,
diluting it with 180 c.c. water and then adding 5 c.c. of bromine, stirring until the
bromine is dissolved. This solution if stored in a*cool dark place will keep about
one week.

The urine to be tested must be free from sugar and albumin and contain less than
i% of urea. Ordinarily the urine must be diluted two to four times to obtain a
specimen containing less than i%. In using this improved Doremus ureometer

FIG. 113. — Doremus- Hinds Ure-
ometer. •

464 APPENDIX

the closed portion of the U tube is filled with the hypobromite solution, and the urine
introduced by allowing it to run in from the side tube by opening the glass cock
arranged for that purpose. After the gas has risen and the instrument has stood
for a short time the readings may be made in grams to the liter, or in percentage.

This urea determination is only a rough clinical one.

Urea Determination by Urease Method. — Put i or 2 c.c. of toluol into each of
two 200 c.c. Erlenmeyer flasks; to one, add exactly 5 c.c. of a specimen and TOO c.c.
of distilled water; to the other, one Urease-Dunning tablet, crushed and dissolved
in about 5 c.c. of water, using a small glass mortar. Rinse the mortar with several
portions of distilled water until about 100 c.c. have been introduced into second flask
and then add exactly 5 c.c. of the urine specimen. Stopper both flasks with cork
and agitate contents. If time is not a consideration, allow flasks to stand at room
temperature, at least eight hours, or use two tablets and digest at 4o°C. for one hour.
Rapid determination may be made by using but i c.c. of urine, two tablets and 100
c.c. of distilled water and digest between 40° and 50° C. for thirty minutes only.

After the elapse of proper time, titrate the two solutions to a distinct pink color
with N/io HC1 and methyl orange. The amount of HC1 required to neutralize
the urease-treated specimen, less the amount required for the control, will give the
urea content, estimated upon its equivalent in ammonium carbonate.

The controls of a large number of determinations made at one time may be ti-
trated, as to existing alkalinity, with N/io HC1 and methyl orange, one after another,
in the same flask, at the time the portions to be examined for urea are prepared in
separate flasks. In such cases, a sufficient number of Urease-Dunning tablets,
for all, may be rubbed up in a mortar with a measured quantity of distilled water
and an aliquot portion, with 100 c.c. of distilled water, added to each of the separate
specimens treated. See urease method for blood.

Gerhardt's Test for Diacetic Acid

Add a few drops of ferric chloride solution to 10 to 50 c.c. of urine as long as a
precipitate continues to form. Then filter and to the filtrate add more ferric chloride
solution. A bordeaux red color shows diacetic acid. The test is sensitive. As a
control to show that the color is not due to drug elimination (antipyrine, salicylates,
etc.) boil a specimen which gave the test for three to five minutes. If the color was
due to drugs it will be obtained with a boiled sample while such treatment drives off
the diacetic acid. In Hurtley's test add 2.5 c.c. HC1 and i c.c. of i% sol. of sod.
nitrate to 10 c.c. urine. Shake and allow to stand two minutes. Now add 15 c.c.
strong ammonia followed by 5 c.c. of 10% sol. ferrous sulphate. The slow produc-
tion of a violet color shows positive test (two hours). Shows i part aceto-acetic
acid in 50,000.

If the urine shows a well-marked Gerhardt reaction it is well to test for /3-oxy-
butyric acid.

The following modification of Lange's test by Hart is a satisfactory one, The
principle involved is the removal of acetone and diacetic acid by heat, then oxidizing
/3-oxybutyric acid to acetone with hydrogen peroxide and then testing for acetone.

Method: Take 20 c.c. of urine, dilute with an equal amount of water and add a
few drops of acetic acid. Next boil in a beaker until the original amount of diluted

APPENDIX 465

urine is reduced to 10 c.c. (originally 40 c.c.). Dilute this evaporated urine with an
equal amount of water, giving us 20 c.c. In each of two test-tubes put 10 c.c. of
this 20 c.c. To one tube add i c.c. of hydrogen peroxide and warm gently, without
boiling, for one minute; then cool. The other tube is left untreated. Next, to each
test-tube add 10 drops of glacial acetic acid and 5 to 10 drops of a freshly prepared
sodium nitroprusside solution and mix. Next carefully overlay each tube with
about 2 c.c. of concentrated ammonia. If 0-oxybutyric acid were present in the
tube treated with the hydrogen peroxide and thereby oxidized to acetone a violet-
red ring will develop at the point of contact while in the untreated tube there will
be no such color ring.

A yellowish-brown ring from the presence of creatinin may show in the untreated
tube. It is well to allow the tubes to stand for three to four hours before finally
reporting the absence of /J-oxybutyric acid. It will probably show 0.2%.

Acetone. — To one-sixth of a test-tube of urine add a crystal of sodium nitro-
prusside. Make strongly alkaline with NaOH. Shake. The addition of a few
drops of glacial acetic gives a purple color to the foam, if acetone is present.

Acetone is naturally present in exceedingly small quantity. It is very much
increased in advanced cases of diabetes mellitus. It is also frequently found in
scarlet fever, typhoid fever, pneumonia, nephritis, and severe anemias. It is often
present after ether or chloroform anaesthesia.

Although the following test can be applied directly to the urine it is preferable
to obtain distillates when possible and test these.

Gunning Test. — To about 5 c.c. of the urine add a few drops of Lugol's solution
and then ammonium hydrate until a black precipitate forms. Allow to stand for
some time, when, if acetone is present, a yellowish precipitate of iodoform crystals
will separate. These should be identified by microscope. Iodoform crystallizes
in some modification of the hexagonal plate, usually a six-pointed stellate form.
If the crystals are not clearly defined, throw precipitate on filter, wash with a little
water, and pour over it a little hot alcohol. To this filtrate add water, drop by drop,
until a precipitate, which will consist of well-defined crystals of iodoform, is obtained.
Examine this by microscope.

Never use an alcoholic solution of iodine in connection with this test.

Diazo Reaction. — To 5 c.c. sulphanilic acid solution (sulphanilic ac. i pt, HC1
50 pts., aq. 1000 pts.) add 2 drops of a 0.5% solution of sodium nitrate. Add an
equal quantity (5 c.c.) of urine. Shake and add quickly 2 or 3 c.c. of ammonium
hydrate. A carmine color, especially in the foam, shows a diazo reaction. If the
reaction is positive, and the mixture is allowed to stand for twenty-four hours, a
precipitate forms, the upper margin of which exhibits a green, greenish-black or
violet zone.

Indican. — Take 10 c.c. urine and treat it with i c.c. of sol. of lead subacetate.
Filter. Of this filtrate take 6 c.c. and treat with an equal amount of Obermayer's
reagent; allow to stand for five minutes then shake gently with 2 c.c. of chloroform.
Obermayer's reagent is strong HC1 containing 2 parts of ferric chloride to the liter
— o.i gram to 50 c.c. of HC1.

A more exact method is to pour off the supernatant acid urine. Wash the chlo-
roform with water, then pour off as much of the supernatant water as possible
and add 10 c.c. of alcohol. A clear blue fluid results.
30

466 APPENDIX

Urobilin. — Urobilin appears in considerable quantity in urine when there is
much destruction of red cells, as in pernicious anaemia, internal haemorrhage, and in
malarial cachexia. The best test is that of Schlesinger. To the unfiltered urine
add an equal amount of a saturated solution of zinc acetate in absolute alcohol.
Shake, add a few drops of Lugol's solution and filter. Fluorescence in the filtrate
shows the presence of urobilin. The degree of blood destruction is indicated by the
intensity of the fluorescence.

Bile Pigments. — A satisfactory test is that of Rosin (Trousseau). Overlay 10
c.c. urine with about 5 c.c. of dilute tincture of iodine (i to 10 of 95% alcohol).
An emerald green ring at the point of contact shows the presence of bile coloring
matter. For Gmelin's lest pass the urine several times through the filter and then
touch the paper with a glass rod which has been dipped in a commercial HNO3.
A green color play shading to blue and violet shows bilirubin.

Bile Acids. — Bile pigments and bile acids usually occur together in urine. A
very delicate and reliable test for bile acids is that of Oliver. Filter a specimen of
urine until it is quite clear. Then make reaction acid provided the urine is not
acid. The urine should be diluted with water until its specific gravity is below
1008. The reagent is made as follows: Peptone 30 grains and salicylic acid 4
grains are dissolved in 8 ounces of distilled water containing % dram of acetic acid.

The solution is filtered until transparent.

For the test add 20 minims of the clear acid urine to 60 minims of the reagent.
A milky turbidity indicates bile acids. If the turbidity disappears on shaking,
the addition of more reagent will cause it to reappear.

URIC ACID IN URINE

Normally we have from 0.2 to 2 grams eliminated in twenty-four hours de-
pending on nature and quantity of protein intake. Nuclein rich foods, as
sweet-breads, liver, etc., greatly increase the amount.

To determine the quantity add to 150 c.c. of the urine 50 c.c. of a reagent which
consists of 500 grams ammonium sulphate, 5 grams of uranium acetate, and 60 c.c.
of 10% acetic acid dissolved, with the aid of heat, in sufficient water (about 700 c.c.)
to make i liter of solution. The mixture of urine and reagent is then filtered. To
134 c.c. of the filtrate (which represents 100 c.c. of the urine), contained in a beaker,
add sufficient ammonium hydrate to make strongly alkaline, and let stand twenty-
four hours. Filter, preferably through hardened filter, wash beaker, and precipitate
thoroughly with 10% ammonium sulphate solution; open filter and with wash bottle
wash precipitate back into beaker, using approximately 100 c.c. of water to do so,
add 15 c.c. of concentrated sulphuric acid, and then immediately from burette add
N/20 potassium permanganate solution until a pink color which persists for thirty
seconds is obtained. The number of cubic centimeters of the potassium per-
manganate solution required is then multiplied by 0.00375; to this result add
0.003 for each 100 c.c. of filtrate and wash solution used. The final result is the
quantity of uric acid in 100 c.c. of urine.

CHLORIDES

These are normally present in quantity corresponding to 10 to 15 grams of sodium
chloride. Diet as well as certain pathologic processes, especially the latter, may cause

APPENDIX 467

marked deviation in quantity and as the deviation is usually very well marked,
a method giving only fairly accurate results is sufficient for their estimation.

Dilute 5 c.c. of the urine, which should be free from albumin, with 50 to 75 c.c.
of distilled water, add 10 to 15 drops of a solution of potassium chromate and then
from a burette add N/io AgNO3 until a very slight pinkish tinge is obtained.
The number of cubic centimeters of AgN03 required, multiplied by 0.00585 will
give the quantity of sodium chloride to which the chlorine in 5 c.c. of urine is
equivalent. Where a considerable degree of accuracy is demanded one should use
either the Arnold or the Harvey modification of Volhardt's method.

FORMALDEHYDE IN URINE AFTER ADMINISTRATION OF UROTROPIN

As formaldehyde fails to appear in the urine of possibly 52% of those taking
urotropin as a geni to-urinary antiseptic in quantities sufficient to inhibit bacterial
growth (1-5000) the usual tests are too delicate. As a practical guide to efficient
breaking up of urotropin the test proposed by Burnam is to be recommended.

To about 10 c.c. urine in a test-tube at body temperature add 3 drops of Y^%
solution of phenylhydrazin hydrochloride and 3 drops of a 5% solution of sodium
nitroprusside. Finally allow a few drops of 20% solution of NaOH to run down
the side of the test-tube and as this diffuses throughout the urine a deep purplish-blue
color, rapidly changing to a dark green becoming lighter green and finally pale
yellow will show if formaldehyde is being excreted in sufficient strength. In the
absence of sufficient formaldehyde a reddish color develops which finally turns to a
light yellow.

In order to obtain effect from urotropin it is necessary to have the urine acid.
This is best accomplished, if an acid reaction is absent, by the administration of
dihydrogen sodium phosphate (acid sodium phosphate). In carrying out Burnam
test albumin in the urine confuses the color reaction. By careful boiling to
precipitate the albumin, then filtering, we avoid confusing colors.

Phenolsulphonephthalein Test for Renal Efficiency

. Geraghty has recently stated that in 35 cases where an autopsy made it possible
to verify the accuracy of this test the lesions revealed at autopsy corresponded
closely with the results of the test. Again in 30 nephrectomies the conditions
found were in accordance with the results of the test. The general opinion of
those who have used the test is that it is more reliable than cryoscopy and far easier
of application. The technic is as follows: One c.c. of the phthalein solution
containing 6 mg. is injected intramuscularly or subcutaneously. The drug can be
bought in ampules ready for use. About twenty minutes before injecting the drug
the patient is given from 200 to 400 c.c. of water to drink. After the injection the
bladder is emptied with a catheter and the time is accurately noted when the urine
which subsequent to the emptying of the bladder and being allowed to drop into a
test-tube containing i drop of a 25% sodium hydrate solution first shows a pinkish
tinge. This is recorded as the time of appearance of the drug in the urine and
normally is about ten minutes. The catheter is then withdrawn and the urine that
is passed in the first hour collected and subsequently that passed in the second hour.

468 APPENDIX

irple-

To each hour's specimen sufficient 25% sodium hydrate is added to give a pui
red colcr and the entire amount is then poured into a liter flask and made up to
looo c.c. A similar treatment is employed for the urine of the second hour. The
amount of drug eliminated in each hour is then determined by a colorimeter.

Cabot has proposed the use of a series of 10 test-tubes containing solutions of
the drug representing from 5% to 50% of the drug dose, each tube containing 5%
more than the preceding one. These comparison solutions may be made up with
the patient's urine obtained at the time of emptying the bladder so that the con-
fusion which may obtain when water is used is avoided. It has recently been pro-

FIG. 114. — Rowntree and Geraghty modification of Hellige Colorimeter.

posed to make the standards with water and use a piece of yellow glass for match-
ing. The urine to be tested made up to 1000 c.c. as previously described is then
poured into a test-tube of similar size and matched.

In normal cases Cabot got 46% of the drug eliminated in the first hour, the
average for the second hour being 17%. The quantity of urine secreted in eithei
hour has no relation to the test, which is the percentage of drug eliminated. In
cases with serious kidney disease the amount of drug eliminated in the first hour
may range from 5 to 12%.

When the question of the kidney involved arises, the urine must be taken by
ureteral catheterization or by a separator.

Dunning has devised a simple inexpensive colorimeter for the drug excretion

APPENDIX

469

determination in place of the more expensive colorimeters such as those of Duboscq
and Hellige. A cut of the apparatus is given. As a rule, I make up my known
standards with urine instead of water as giving better comparison and pour a darker
known solution from a graduated cylinder into a Nessler jar as described under
blood-sugar estimations.

FIG. 115. — Dunning colorimeter.

F— CHEMICAL EXAMINATION OF GASTRIC CONTENTS

The test breakfast ordinarily used is that of Ewald (one shredded wheat biscuit
or two small pieces of toast with 400 c.c. of water is what is usually given). This
Ewald breakfast is a low-grade stimulant to acid production. It is given in the
morning on an empty stomach. If at supper, the night before, the patient partake
of raspberry jam the finding of the characteristic seeds in the stomach contents
the next morning would be evidence of lack of motor activity. The Fischer meal
which contains a 4-ounce Hamburg steak in addition to the water and toast of the
Ewald is withdrawn after three hours. v

The stomach tube is more easily passed if it be thoroughly chilled in ice water
without the use of any lubricant.

The stomach tube should be passed one hour after the Ewald breakfast and if
more than 50 c.c. of fluid be obtained it indicates stasis or hypersecretion.

Free HCL— Filter the gastric contents and test first for free HC1. The most
reliable and sensitive test is that of Gunsberg. The reagent, which should be
freshly prepared, consists of phloroglucin 3 grams, vanillin i gram, and absolute
alcohol 30 c.c. By mixing 2 drops of gastric juice and an equal quantity of Gunsberg
reagent in a small porcelain dish and carefully heating above a flame we obtain a
carmine red color if free HC1 be present. A water-bath is preferable.

The Gluzinski test is of value in the diagnosis of gastric cancer. In this, three

470 APPENDIX

tests for free HC1 are made, one from gastric contents and washings taken in the
morning before taking any form of food. Then give the albumin of 2 hard boiled
eggs, finely minced in 150 c.c. water. One hour later remove the contents and ex-
amine for free HC1. Then give a meal of broth, lightly broiled steak, mashed
potatoes and bread and three hours later remove contents with stomach tube and
test again for free HC1.

For lactic acid a modification of Strauss' method is quite satisfactory. Shake,
in a test-tube, 5 c.c. of gastric contents with 20 c.c. of ether, allow to settle and
pour off 5 c.c. of the supernatant ether into another test-tube. To this ether add
20 c.c. of water and 2 drops of a i to 9 solution of ferric chloride and shake well.
The presence of i% of lactic acid will give an intense greenish color.

Total Acidity. — Having determined the presence or absence of free hydrochloric
or lactic acid, we should make a quantitative test of the various determinations of
the acidity of gastric juice (a modified Topfer test). These are: i. Free HC1. 2.
Combined HC1. 3. Acid salts, and 4. Total acidity.

To 10 c.c. of filtered gastric contents, in a beaker, add 3 drops of dimethyl-amido-
azo-benzol solution (a 1/2% solution in 95% alcohol). In the presence of "free
HC1 the fluid becomes a rich carmine pink.

After reading the burette run in N/io NaOH solution until the pink color is
discharged and a light yellow color is obtained. This reading multiplied by 10
gives the' amount of free HC1 in degrees, a degree corresponding to i c.c. N/io
NaOH. Next add 6 drops of a 1/2% alcoholic solution of phenolphthalein to the
light yellow fluid in the beaker. Again titrating the same preparation we add
N/io NaOH until a faint but distinct pink color is produced. The number of
cubic centimeters added for the free HC1 plus the number to give the pink color
when multiplied by 10 gives the total acidity in degrees. (For example: 2.5 c.c.
N/io NaOH used to obtain yellow color — 2.5X10 = 25 or acidity due tofreeHCl.
After adding the phenolphthalein, 4 c.c. N/io NaOH required to produce pink
color — 4+2.5X10 = 65 or total acidity in terms of acidity. This means that it
would require 65 c.c. N/io NaOH to neutralize 100 c.c. of gastric juice. A total
acidity of 60 is about normal. To obtain percentage in HC1 multiply by 0.00365;
thus, 65X0.00365 = 0.23% HC1.)

Having determined the total acidity add 3 c.c. of 10% neutral calcium chloride
solution to the gastric contents already in the beaker. As a result of the formation
of acid calcium phosphate the pink color is discharged. Again add N/io NaOH
from the burette until the pink color is restored. The number of cubic centimeters
used gives the amount of acid salts present.

From the figures for the total acidity subtract the sum of that for free HC1 and
for acid salts and the remainder will give the acidity due to combined HC1.

WOLFF AND JUNGHANS' TEST

This is an important test as distinguishing benign achylias from those associated
with malignant disease of the stomach. Of course, where the carcinoma originates
in the lesion of an ulcer or when small or in the region of the pylorus there may not
be an achylia, free HC1 being present.

While interpretation of increased albumin in those cases of carcinoma showing

APPENDIX 471

free HC1, owing to digestive action, would be difficult, this is less so in the typical
case with its absent free HC1. To carry out the test it is best to give the patient
only a light supper and follow this with mild cathartic the same evening. Give
the Ewald test breakfast the next morning and withdraw contents in one hour. Fil-
ter the contents and of the nitrate deposit i c.c., 0.5 c.c., 0.25 c.c., o.i c.c., 0.05 c.c.,
and 0.025 c.c. in six test-tubes. Make up contents of each tube to 10 c.c. with
distilled water. The dilutions will be from i to 10 in the first tube to i to 400 in the
sixth tube. After mixing the tubes superimpose i c.c. of the reagent on each tube.
The reagent is phosphotungstic acid 0.3 gram, concentrated HC1 i c.c., 96% alcohol
20 c.c., distilled water to make 200 c.c.

Normal gastric juice gives a turbid albumin ring in the first two or occasionally
in the third (i to 40) tube. Turbidity in the i to 100 or over is suggestive of car-
cinoma.

The increase of albumin content in malignant achylias is thought to be due to
the action of a specific ferment capable of acting on the protein content of the
meal forming the soluble albumin we test for.

PEPSIN

The determination of the quantity of pepsin is rather troublesome by the older
methods, and in consequence a simple method, which also uses a protein, will be given.
The protein used is edestin, a globulin obtained from hempseed. Dissolve o.i
gram of the edestin in 100 c.c. of 0.1% HC1. (This is most quickly done by
sprinkling the edestin over the surface of the acid.) In each of six small test-tubes
place exactly 2 c.c. of the edestin solution (thus giving 2 mg. of the substance
to each tube). Dilute i c.c. of stomach contents to 20 c.c. with water. To the above-
mentioned test-tubes are added, respectively, 0.2, 0.4, 0.6, 0.8, and i c.c. of the
diluted sample, the last test-tube being left as a blank. Shake each test-tube so
that its contents will be mixed. Allow the tubes to stand for thirty minutes. Then
to each tube add 0.3 c.c. of a saturated solution of NaCl. In those tubes which do
not contain sufficient pepsin to digest 2 mg. of edestin a white cloud or precipitate
will be produced while all others remain clear. (The blank or sixth tube must give
a copious precipitate or the edestin solution is worthless.) The tube containing the
smallest quantity of the dilute sample and giving no cloud or precipitate is selected.
Then by the formula 2 x dilution divided by the volume of dilute sample present is
obtained the number of milligrams of edestin that will be digested by i c.c. of the
undiluted sample. This quantity normally is said to be 100. If all but the sixth
tube failed to give the cloud or precipitate a greater dilution of the sample must be
made and the test repeated. The edestin solution rapidly decomposes and must
be made fresh when needed.

Mett's Method for Quantitative Estimation of Pepsin. — Capillary glass tubes,
i or 2 mm. in diameter are filled with white of egg, and after plugging one end with
bread crumbs the tubes are placed in water at 97° or 98°C. for five minutes. The
tubes are sealed with plasticine or sealing wax. For the test dilute i c.c. of gastric
juice with 15 c.c. N/20 HC1 and put some into a test-tube. Then file off two 2 cm.
columns of the capillary tube with coagulated egg albumin. Place test-tube with its
albumin tubes in incubator for twenty-four hours. Then measure the digested ends

472

APPENDIX

of the two capillary tubes and take average. The square of this figure gives
number of units of pepsin in the i to 16 dilution of gastric juice and when multi-
plied by 1 6 the number of units in i c.c. of gastric juice. Normal variation is
8 to 100 units.

G— CHEMICAL EXAMINATION OF DUODENAL JUICE

The general discussion of the subject of the examination of the duodenal juice is
taken up in Part IV. As stated there the lipase tests are rather unreliable so only
tests for amylase (amylopsin) and protease (trypsin), as recommended by Myers
and Fine are given.

Wohlgemuth Method for Amylase. — Place 5 c.c. of i% soluble starch solution in
each of six small test-tubes. Tube I serves as a control, and to each of the other five
tubes add 0.05, o.i, 0.25, 0.5 and i.o c.c. of the duodenal juice diluted one half with
distilled water.

Place in 37°C. incubator for one-half hour, add cold water to almost fill the tubes
and then several drops of N/io iodine. The positive tube will show an entire ab-
sence of blue color. The amylase strength is represented by the number of cubic
centimeters of starch solution i c.c. of undiluted juice can digest. If i c.c. of un-
diluted duodenal juice digests 5 c.c. of starch solution the amylase strength is 5; if
o.i, 50. The average normal is about 50.

Gross Casein Method for Trypsin. — Put in each of six test-tubes 5 c.c. of o.i % solu-
tion of casein in o.i % sodium carbonate solution and add to each one, except tube I,
which serves as control, the same amount of diluted duodenal juice as given above
for amylase. Incubate for fifteen minutes at 38°C. and acidify with a few drops of
dilute acetic acid. The activity is calculated as for amylase and is normally from
4 to 10. White makes a i% dilution of the duodenal fluid with water and puts 10
c.c. of 0.1% casein solution in each tube. The tube with o.i c.c. of diluted fluid
would therefore be a i to 10,000 dilution and that with i c.c. a i to 1,000. Nor-
mally the i to 10,000 tube remains cloudy. Clouding of the i to 1,000 tube would
show marked lowering of tryspin.

H— DISINFECTANTS AND INSECTICIDES

By disinfection is meant the destruction of injurious bacteria.

Sterilization is where all living things are destroyed.

Germicides are substances which kill bacteria while antiseptics are those which
are inimical to the growth of bacteria.

Formalin is antiseptic in 1-50,000 dilution but germicidal only in 1-20.

Deodorants may or may not be antiseptic or germicidal. An insecticide may
or may not be a germicide and vice versa.

In disinfection we must consider

(1) Strength of solution. It must always be kept in mind that the strength of
a germicide solution when added to an equal amount of material to be disinfected
is reduced in strength one-half. Thus i pint of a 5%'comp. cresol solution added to
i pint of faecal material has only a 2^% disinfecting effect.

(2) Time of application. A common mistake is to consider a few minutes as suffi-
cient for contact of germ-containing material with the disinfectant. In the faeces-

APPENDIX 473

cresol mixture above noted that action of the disinfectant should continue at least
one hour before emptying the vessel.

(3) Nature of medium in which disinfectant acts. Bacteria are much more re-
sistant to germicide agents when contained in solution rich in organic matter than
when suspended in pure water. Thus Liq. Cresol. Comp. has a phenol coefficient
of 3 without organic matter and only 1.87 with organic matter.

(4) Temperatures. Disinfecting solutions show greater power as the temperature
rises, and act less efficiently in the cold. A body temperature (3Q°C.) is a good
one.

By Coefficient of Inhibition we mean time and concentration necessary to prevent
development of bacteria.

By Inferior Lethal Coefficient we mean time and concentration necessary to kill
nonspore-bearing bacteria.

By Superior Lethal Coefficient we mean time and concentration necessary to
kill spore-bearing bacteria.

U. S. Hygienic Laboratory Phenol Coefficient. — In determining the strength of a
disinfectant it is compared with that of phenol (commercial carbolic acids vary in
their phenol content). If more powerful than phenol the coefficient will be greater
than i. In the U. S., disinfectants are rated according to the "Hygienic Laboratory
Phenol Coefficient."

The determinations when organic matter was not present, were conducted as
follows :

"The experiments were done at a temperature of 2o°C. maintained by means of a
water-bath. The quantity of each dilution of disinfectant and phenol used in each
experiment was 5 c.c. The culture used was B. typhosus, twenty-four-hour
extract broth, filtered. The seeding tubes containing the disinfectant dilutions and
the filtered broth culture of B. typhosus were placed in the water-bath and allowed
to reach the temperature of 2o°C. before starting the experiment.

" The seeding tubes were inoculated successively every fifteen seconds with o.i c.c.
of the typhoid culture. Each tube was gently shaken after it was inoculated. At
the end of each two and one-half minute period, for fifteen minutes, plants were
made from the seeding tubes into tubes of extract broth. The medium used was
standard extract broth having a reaction of +1.5. The quantity of broth in each
tube was approximately 10 c.c.

"The tubes were incubated at 37°C. for forty-eight hours, at the end of which
the results were recorded.

" To determine the coefficient, the figure representing the degree of dilution of the
weakest strength of the disinfectant that kills within two and one-half minutes is
divided by the figure representing the degree of dilution of the weakest strength of
the phenol control that kills within the same time. The same is done for the
weakest strength that kills within .fifteen minutes. The mean of the two is the
coefficient.

" When the determinations were made as to efficiency in the presence of organic
matter a stock solution of 10% peptone and 5% gelatin in water was made up and
of this i c.c. was put in a tube and then inoculated with o.i c.c. of typhoid culture.
Then 4 c.c. of the varying phenol and disinfectant dilutions were added and the
tests conducted as above."

474 APPENDIX

Disinfectants may be (A) Physical, (B) Gaseous, (C) Chemical

(A) Of the physical disinfectants we have

(1) Sunlight. The red and yellow rays practically inert. The violet and ultra
violet most active. Direct sunlight kills plague bacilli in less than one hour —
typhoid bacilli in six.

(2) Burning. Very efficient but expensive.

(3) Boiling. Especially in carbonate of soda solution for about one hour is a
very efficient disinfectant. Nonspore-bearing bacteria are killed almost instantly
by a boiling temperature. One must remember that the boiling temperature is
lower at mountainous elevations.

(4) Steam. Extremely efficient. The condensation of the steam on the object
to be sterilized gives off latent heat and produces a vacuum.

(B) Of the gaseous disinfectants we have the very efficient germicide formal-
dehyde gas and the weakly gerrnicidal, but potent insecticide, sulphur dioxide.

Formaldehyde gas is practically valueless as an insecticide.

Bromine, chlorine and hydrocyanic acid gas have a certain degree of efficiency
but are not of practical application. Hydrocyanic acid gas is especially dangerous
on account of its extreme toxicity.

(i) Formalin. — This is a 40% solution of formaldehyde gas, but is as a rule
of less strength from evaporation or otherwise. Formaldehyde is efficient as a
surface disinfectant when the temperature is above 5o°F. and the air contains at
least 60% of moisture. It is not efficient in cold dry rooms. Owing to its lack
of penetrating power it is not efficient for the disinfection of mattresses, or similar
articles. To prepare a room for disinfection we must measure the cubic space to
ascertain the necessary amount of formalin to use and stuff up or better paste
up with newspaper all cracks and openings.

In the production of formaldehyde gas the more expensive autoclaves and lamps
have largely been replaced by the simple formalin permanganate method. In this
one pours 500 c.c. of formalin on 250 grams of potassium permanganate for each
1000 cubic feet with six to twelve hours' exposure.

In employing this method, take a pan partly filled with water. Place in this
a second metal or glass receptacle containing the permanganate. Then pour the
formalin on the permanganate crystals. The gas is generated in great amount in
a few seconds. The receptacle containing the permanganate and formalin should
be large enough to contain 10 times the volume of formalin, as there is a tendency
for the mixture to foam over the sides of the dish.

Another practical method is the fonnalin-sheet-spraying one. The formalin
(40%) should be sprayed on sheets suspended in the room in such a manner that
the solution remains in small drops on the sheet. Spray not less than 10 ounces
of formalin (40% formaldehyde) for each 1000 cubic feet. Used in this way a
sheet will hold about 5 ounces without dripping or the drops running together.
The room must be very tightly sealed in disinfecting with this process and kept
closed not less than twelve hours. The method is limited to rooms or apartments
not exceeding 2000 cubic feet. The formalin may also be sprayed upon the walls,
floors, and objects in the room.

Paraform Lamps. — For single rooms the use of the paraform lamp is quite con-
venient. Special lamps can be obtained to burn the paraform tablets or a pint

APPENDIX 475

tincup will suffice for the heating of i ounce of paraform. The lamp or alcohol
flame under the receptacle must not be high enough to ignite the paraform which
burns readily and in so doing does not give off formaldehyde gas. One ounce of
paraform is sufficient for a space of 500 cubic feet. One can dissolve 2 ounces of
paraform in 8 ounces of boiling water and then pour .this over 4 ounces of potassium
permanganate in a 2-gallon pail.

N. Y. Health Department Method.— After a prolonged series of tests the N. Y.
Department of Health gave preference to the following formula:

Paraformaldehyde 30 grams, potassium permanganate 75 grams, water 90
grams. The chemicals are mixed in a deep quart pan and the water is added and
the mixture stirred. The evolution of gas is slow in starting but is complete in
five to ten minutes.

It was found that 87% of the gas was evolved and the quantities given above
suffice to disinfect 1000 cubic feet in four hours. It is well to put the small pan
containing the chemicals in a larger one to prevent danger of fire and soiling of the
floor by the frothing of the mixture.

Sulphur Dioxide. — Sulphur dioxide is fairly efficient, but requires the presence
of moisture. It is only a surface disinfectant and is lacking in penetrating proper-
ties. An atmosphere containing 4.5% can be obtained by burning 5 pounds of
sulphur per 1000 cubic feet of space. This amount requires the evaporation or
volatilization of about i pint of water. Under these conditions the time of ex-
posure should be not less than twenty-four hours for bacterial infections. A shorter
time will suffice for fumigation necessary to kill mosquitoes and other vermin. Dry
sulphur dioxide produced by burning 2 pounds of sulphur for each 1000 cubic feet
of space will" answer for this purpose. An exposure of from two to three hours is
sufficient.

The sulphur may be burned in shallow iron pots (Dutch ovens), containing not
more than 30 pounds of sulphur for each pot, and the pots should stand in vessels
of water. The sulphur pots should be elevated from the bottom of the compart-
ment to be disinfected in order to obtain the maximum possible percentage of com-
bustion of sulphur. The sulphur should be in a state of fine division, and ignition
is best accomplished with alcohol (special care being taken with this method to
prevent damage to cargo or vessel by fire), or the sulphur may be burned in a special
furnace, the sulphur dioxide being distributed by a power fan. This method is
peculiarly applicable to cargo vessels.

Liquefied sulphur dioxide may be used for disinfection in place of sulphur dioxide
generated as above, it being borne in mind that this process will require 2 pounds
of the liquefied gas for each pound of sulphur, as indicated in the above paragraphs.

Sulphur dioxide is especially applicable to the holds of vessels or to apartments
that may be tightly closed and that do not contain objects that would be injured
by the gas. Sulphur dioxide bleaches fabrics or materials dyed with vegetable or
aniline dyes. It destroys linen or cotton goods by rotting the fiber through the
agency of the acids formed. It injures most metals. It is promptly destructive of
all forms of animal life. This property renders it a valuable agent for the extermi-
nation of rats, insects, and other vermin. Sulphur dioxide is a germicide only in the
presence of moisture, and even then will not kill spore-bearing organisms. If 'cloth-
ing is washed immediately after sulphur disinfection the rotting effect will be greatly

476 APPENDIX

lessened. If used in spaces containing machinery all metal parts should be coa
with vaseline.

CHEMICAL SOLUTIONS

Bichloride of mercury is usually sold in the form of antiseptic tablets. As a
disinfectant. for the infectious diseases it is usually used in a strength of i-iooo.
The solution should be made in a wooden or earthenware vessel. As bichloride forms
inert albuminates it should not be used in the disinfection of sputum, faeces or any
albuminous excreta. It must be remembered that bichloride is a mordant so that
any stains in soiled clothing will remain permanent. For disinfection of clothing
the material should be left in i-iooo bichloride for one hour. Dishes for food
should never be disinfected in bichloride on account of the danger from poisoning.
Floors and walls may be disinfected with i-iooo bichloride applied with a mop.
Allow the solution to dry on the floor or walls.

Formalin. — A 5% solution of commercial formalin in water (50 c.c. formalin
950 c.c. water) makes a satisfactory disinfectant for soiled clothing. It is also
valuable for albuminous material. The disinfectant must act in a strength of 5%
so that if i pint of fasces is to be disinfected we should add i pint of a 10% formalin
solution and allow it to act for one hour.

Carbolic Acid. — It is soluble in water to the extent of about 5% and in such
strength it is an efficient disinfectant. The solution should be made with hot water.

In standardizing disinfectants carbolic acid is used as the standard. It how-
ever is expensive and there is often difficulty in making up satisfactory solutions.
More efficient and more convenient is the Liquor cresolis comp. U. S. P. This may
be prepared by mxing up equal parts of cresol and soft soap as noted on page 13.
This has a value according to tests made in the Hygienic Laboratory of 3, making
it in tests without organic matter three times as efficient as carbolic acid. Under
similar conditions lysol had a value of 2.12, creolin 3.25 and trikresol of 2.62.

Equal parts of a 5% solution of Liq. Cresol. Comp. and the fasces, urine or
sputum to be disinfected is satisfactory for disinfection provided the mixture is
allowed to stand for one hour. Here we would have the effect of a 2^% solution.
Liq. Cresol. Comp. (5%) is an excellent disinfectant for contaminated bedclothing,
etc. It is also most suitable for the disinfection of floors and walls.

Sulphate of Copper. — This salt has a remarkable effect on certain species of algae
so that in strengths of i to 1,000,000 it is destructive. In i to 400,000 it will kill
typhoid bacilli in twenty-four hours in water that is not too full of organic matter.

Hydrogen Dioxide. — A 2% solution will kill anthrax spores in three hours. It
is useful in treatment of anaerobic infections, as gas bacillus ones.

Chinosol. — This is a derivative of quinoline, a coal-tar product. It is a yellow
powder readily soluble. It does not coagulate albumin and leaves no stain on
clothing. It is efficient in a strength of i to 500 or i to 1000.

Lime. — It must be remembered that air-slaked lime is inert as a disinfectant.
For disinfecting faeces freshly prepared milk of lime is excellent. It is made by
mixing unslaked lime with four times its volume of water. An equal quantity
should be added to the faeces to be disinfected.

Chlorinated Lime. — This can be purchased in air-tight containers and when the
package is opened it should give off a powerful oder of chlorine.

APPENDIX 477

For a working disinfectant solution add i pound to 4 gallons of water. This
is satisfactory for mopping floors and for disinfecting faeces, sputum and urine,
equal parts of the excreta and disinfecting solution being mixed and allowed to stand
for one hour. For disinfection of drinking water one teaspoonful of chlorinated
lime to i pint of water makes a stock disinfectant. For use one teaspoonful of
this stock solution is added to .2 gallons of the drinking water to be disinfected.
Let stand at least one-half hour.

Eusol. — A solution containing 0.5% hypochlorous acid and known as eusol has
been highly recommended in the treatment of gas gangrene wounds. To make it
put 27 grams chlorinated lime (bleaching powder) in a Winchester quart flask and
cover with a liter of water. After thorough shaking add 27 grams of boric acid.
After shaking the mixture should stand for a few hours and then be filtered through
cotton wool. The clear solution is eusol. It must be kept in tightly closed bottles.

INSECTICIDES

The following notes are taken chiefly from the U. S. P. H. Service directions.

Sulphur Dioxide — obtained as described above — destroys all animal life.

In the case of vessels, when treated for yellow fever infection, the process shall
be a simultaneous fumigation with sulphur dioxide, 2% volume gas, and two hours'
exposure in order to insure the destruction of mosquitoes.

In the case of vessels when treated for plague the process with sulphur dioxide
shall be as follows:

Without cargo: The simultaneous fumigation with sulphur dioxide gas not less
than 2% for six hours' exposure.

With cargo: Fumigation with sulphur dioxide gas, 4%, six to twelve hours'
exposure, according to stowing.

Infected vessels may require partial or complete discharge of cargo, and frac-
tional fumigation for efficient deratization.

Pyrethrum. — The fumes of burning pyrethrum may be used to destroy mos-
quitoes in places where there are articles liable to be injured by the use of sulphur.

Four pounds per 1000 cubic feet space for two hours' exposure will kill all, or
practically all, of the mosquitoes but precautions should be taken to sweep up and
destroy any that may have escaped.

Pyrethrum stains walls, paper, etc.

The Oxides of Carbon, as used at Hamburg, are efficient to destroy rats but do
not kill fleas or other insects. They are obtained by burning carbon, coke, or char-
coal, in special apparatus, and the gas as produced consists of about 5% carbon
monoxide, 18% carbon dioxide, and 77% nitrogen.

Twenty kilos of carbon, coke, or charcoal are used for every 1000 meters of
space. The gas is allowed to remain in the ship for two hours and from seven to
eight hours are allowed for it to leave it. This is about equivalent to i K pounds
of carbon (coke) to 1000 cubic feet of air space. As this gas is very fatal to man and
gives no warning of its presence, being odorless, a small amount of sulphur dioxide
should be added to give warning of its presence. As it does not kill fleas it cannot
be depended on for complete work, where there is evidence of plague among rats
on the vessel, as the infected fleas would infect the rats coming aboard after the
deratization.

478 APPENDIX

The articles named as disinfectants which can obviously destroy animal life can
be used for that purpose when applicable, as steam for bedding, fabrics, etc. For-
maldehyde is not applicable for this purpose.

Pulicides. — For fleas the best insecticides are (i) crude petroleum (fuel oil) which
is at times called Pesterine, (2) an emulsion of kerosene oil made as follows: kerosene
20 parts, soft soap i part and water 5 parts. The soap is dissolved in the water by
aid of heat and the kerosene oil gradually stirred into the hot mixture.

For cockroaches there is nothing so good as sodium fluoride. By sprinkling the
powder about the haunts of the cockroaches they are gotten rid of in a few days.

Pediculicides . — Owing to their great importance in transmitting typhus fever
and relapsing fever the destruction of human lice is a vital consideration.

While the body louse is the important transmitting agent, the head louse and pos-
sibly the crab louse should also be destroyed.

The subject of pediculosis has been much discussed on account of its importance
among the troops in the European war. In Shipley's book on the "Minor Horrors
of War" the following methods of destroying lice are given. Fernet gives the follow-
ing instructions for the body louse.

1. All body and bed-linen and clothes should be baked or sterilized by boiling.

2. Unguentum staphisagriae should be applied to neck-bands of vests and shirt in
the region of the neck.

3. Alkaline baths to soothe the irritated skin.

Flowers of sulphur sprinkled in the bed and in the clothes is very useful.

Major Lelean recommends: A 2-inch loose-woven bandage is made into a
tubular bag. Into this bag 2 teaspoonsful of the following powder is poured and
evenly distributed.:

Naphthalene 96%

lodoform 2%

Creasote 2%

The bag is then tied round the waist, and it is said that all lice are killed within
twenty-four hours.

Moor head advises the dusting of flowers of sulphur on the clothes but many re-
ports indicate the inefficiency of this method.

For head lice Fernet recommends :

1. Prevention: hair to be kept close cropped and clean.

2. For the nits: wipe them off with a solution of i in 30 carbolic acid.

3. For the lice themselves: Unguentum hydrargyri ammoniat. dil. (gi. xto i oz.),
or any fatty, sticky body well rubbed into the back of the head. Paraffin lamp-oil
(kerosene) also good, but not to be used near a naked flame or light.

Blanchard considers camphorated alcohol or warm vinegar containing i to 1000
of corrosive sublimate as useful for head lice. He also suggests the fumigation of
clothes with tobacco as valuable for body lice.

Castellani and Jackson have gone most extensively into the matter of louse des-
truction. Their conclusions are as follows: i. In regard to solid and liquid insecti-
cides, the substances which have been found to be deleterious to body-lice are, in
the order of their efficiency: Kerosene oil, vaseline, guaiacol, anise preparations,
iodoform, lysol, cyllin and similar preparations, carbolic acid solution, naphthaline,
camphor.

ran

APPENDIX 479

Pyrethrum has a very feeble action on lice, while boric acid, sulphur, corrosive
sublimate, and zinc sulphate, when used in powder form, have apparently no action
whatever. As regards bedbugs, kerosene oil is the best insecticide. Next to it
comes guaiacol, one of the most active drugs of those tried.

2. For use against lice on a large scale, as among troops and prisoners, perhaps the
best insecticide powder is naphthaline. This substance has a lower licecide action
than kerosene oil, guaiacol, iodoform and anise preparations, but it has less
displeasing odor than the first three named. In stored blankets and clothing
it is also practicable and of use, as frequently lice are found upon the clothing
and blankets stored through the summer.

Medical Director H. G. Beyer, U. S. N., gives the following description of the
method of louse destruction employed by Lenz at the prison camp at Puchheim.
On the arrival of a large transport of prisoners, the clothes they wear on their persons
are, first of all, subjected to steam-disinfection, whereby a great mass of lice, includ-
ing eggs, are destroyed. The hair on the bodies of the men is then shorn with a
machine, and they are lathered over with soft soap and put into a bath. The now
disinfected clothes are put on again and treatment with naphthalin is begun under
the supervision of the sanitary police. Every person, at bedtime, has a handful of
finely powdered naphthalin put into his clothes, introduced through the opening
at the neck. He is made to sleep that night with all his clothes on him. The body
heat causes the naphthalin to evaporate, the vapors killing not only the remaining
lice, but also most of the remaining eggs. If this treatment is repeated twice more,
regardless of whether living lice are found or not at the time, a thorough and com-
plete louse disinfection will be assured. Lenz does not mention any disagreeable
effects of the naphthalin vapors on the men themselves. When dealing with trans-
ports of smaller numbers of individuals, Lenz states that he has succeeded even
without the employment of steam-sterilization of the clothes. The advantages
of the naphthalin method as used by him are given as follows:

1. That it is cheaper than any other method, its cost per person being \y± cents.

2. That it does not interfere with the service efficiency of the men.

3. That it requires neither special apparatus nor places.

4. That it does not injure clothing.

5. That it is absolutely noninjurious to the health of the men.

Raticides.— For exterminating rats and in this way secondarily the rat-fleas, be-
sides the ordinary poisons such as As, P, etc. Rucker has recommended a poison
composed of plaster of Paris, 6 parts, pulverized sugar i part and flour 2 parts. This
mixture should be exposed in a dry place in open dishes. To attract the rats the
edge of the dish may be smeared with the oil in which sardines have been packed.

Larvicides. — Wise and Minett report good results from the use of crude carbolic
acid as a larvicide for mosquitoes. They added about i teaspoonful for each 2
cubic feet of water in the pool. Of course, the ordinary method for destroying mos-
quito larvae is by covering the surface of the water in the cistern or pool with a layer
of petroleum.

I— ANATOMICAL AND PHYSIOLOGICAL NORMALS

In examinations in the pathological or chemical laboratory the following may be
considered approximately as normal findings:

480 APPENDIX

A. Anatomical normals of adult males. Averages.

Adrenals. The two adrenals together weigh about % ounce (10 grams) or
grams each.

Brain. Weight 50 ounces (1400 grams).

Heart. Weight, n ounces (310 grams). Length, 5 inches (125 mm.). Breadth,
3^ inches (87 mm.). Thickness, wall, left ventricle, ^ inch (9 mm.), right ven-
tricle, Ko inch (2.5 mm.). Circumference, mitral orifice, 4% inches (10.5 cm.).
Circumference, tricuspid orifice, 5 inches (12.5 cm.). Circumference, aortic orifice,
3*4 inches (8 cm.). Circumference, pulmonary orifice, 3% inches (9 cm.).

Intestines. Small intestine, 24 feet. Large intestine, 5 feet. Duodenum, 10
inches.

Kidneys. The average weight of both kidneys is about n ounces (310 grams).
The left kidney weighs about 6 ounces (160 grams), and the right about 5^ ounces
(150 grams). The thickness of cortex is about ^i inch.

Liver. Weight 45 to 50 ounces (1600 grams). Greatest transverse diameter, 9
inches (25 cm.). Greatest antero-posterior diameter, 4^ inches (n cm.).

Lungs. Combined weight 39 ounces (uoo grams). Right lung about 2 ounces
heavier than left.

Pancreas. The weight of this organ, like the spleen, is quite variable. An
average weight would be about 2^ ounces (70 grams).

Spleen. Weight 6 ounces. (175 grams). Length 5 inches (12.5 cm.). Breadth,
3 inches (7.5 cm.). Thickness, i% inches (3 cm.).'

Testes. The left testicle is slightly larger than the right and its weight is about
% of an ounce (20 to 24 grams).

B. Physiological Normals.

Blood.

Specific gravity 1041 to 1067

Specific gravity (blood-serum) 1026 to 1032.

Haemoglobin 14%.

Serum albumin 4.52%.

Paraglobulin 3.10%.

Fibrinogen 0.42%.

Glucose 0.84 to 0.1%.

Sodium chloride 0.65%.

Cholesterol 0.15%.

Non-protein N 25 to 35 mg. in 100 grams blood.

Urea N 1 2 to 23 mg. in 100 grams blood.

Uric acid i to 2 mg. in 100 grams blood.

Creatinin /. . . . i to 2 mg. in 100 grams blood.

Cerebro-spinal fluid.

Specific gravity 1007 to 1010.

Urea o.oi to 0.05%.

Total proteid 0.025% (Albumin not present normally).

Pressure of fluid 5 to 7.5 mm. mercury or 60 to 100 mm

water.

..

APPENDIX 481

Faeces.

Amount in twenty-four hours on ordinary mixed diet, no to 170 grams (Solids

25 to 45 grams).

Amount in twenty-four hours on vegetable diet up to 350 grams (Solids 75

grams).

Average daily output, moist faeces (Hawk) 100 grams.
Gastric juice.

One hour after Ewald breakfast.

Quantity 40 to 50 c.c.

Total acidity 40 to 60 (0.15 to 0.22%).

FreeHCl 20 to 60 (0.05 to 0.2%).

Combined % to 3 (o.oi to 0.1%).

Contents of fasting stomach (residuum) 20 to 50 c.c. (Rehfuss tube).

Total acidity of residuum 30.

Free HC1 acidity 19.

Respiration.

Composition of alveolar air: Oxygen, 14.5%; Carbon dioxide, 5.5%; Nitrogen

80%.

Some give CO2 alveolar content as from 3.7 to 5.5 volume per cent., or CO2

tension of alveolar air as from 35 to 40 mm.

Air hunger in diabetes or chronic nephritis only begins when CO2 tension has

fallen to 20 or 25 mm.
Urine. Amount (American male) 1 200 c.c.

Specific gravity 1.015 to 1.025

Urea 2.3% (35 grams in twenty-four hours) about

90% of total nitrogen.

Uric acid 0.05% (.75 grams in twenty-four hours).

Creatinine 0.07% (i gram in twenty-four hours).

Ammonia 0.04% (.7 gram in twenty-four hours).

NaCl 1.1% (!6-5 grams in twenty-four hours).

INDEX

Abbe" condenser, 5
Abderhalden technique, 195

Bronfenbrenner's, 196
Abscess, bacteria in, 420
Acanthia lectularia, 268, 344, 348, 379

rotundata, 348
Acarina, 334
Acidosis, 218, 402, 456

tests for, 220, 456, 462
Acartomyia, 376
Acetone, for sections (see tissue), 445

in urine, 465
Achromia, 223
Acid-fast bacteria, 90, 91

staining, 41
Acid proofing, 13
Actinomycosis (see Discomyces), 143,

392, 420
^Edinae, 374
Agar, egg, 27

gelatin, North, 28

glucose, 27

glycerine, 27

nutrient, 26

plating, 49

Vedder's starch, 27
Agglutination, macroscopical, 169

microscopical, 168
Ainhum, 438
Air, bacteriological examination of,

. 159

Albumin in urine, 457
Albumin in sputum, 392
Albumose in urine, 458
Aldrichia, 373
Alexin, 165
Allergy, 194

Alveolar air tension, 220, 456
Ambard index, 402

Amboceptor, 164

native in sheep cells, 178
Ammonia in urine, 462
Amoebae, 252
Amcebae, of intestines, 252

of mouth, 258

staining of, 46, 256
Anaemia, aplastic, 224

infantum, 241

pernicious, 236

primary, 235

secondary, 237
Anaerobes, 78

Buchner method, 79

combination method, 80

cultivation of, 78

Liborius method, 79

Noguchi method, 36

roll culture, 50

Tirozzi's method, 78

Vignal method, 80

Wright method, 80

Zinsser method, 80
Anaerobiasis, 55
Analogy, 248
Anaphylaxis, 192
Anaphylactic shock, 193
Anaphylactine, 194
Anaphylatoxin, 194, 196
Anatomical normals, 480
Ancylostoma duodenale, 313, 32 5, 411,

422

Anginas, 61, 387
Anguillula, 314, 396
Animal inoculations, 55, 167, 176
Animal parasites, general classifica-
tion, 243, 245

key to, 245

mounting of, 450

483

484

INDEX

Animal parasites, nomenclature in,
246

preservation of, 450
Anisocytosis, 223
Anlage, 248
Anophelinae, 373
Anthomyia, 326, 354, 361
Anthrax, 75

symptomatic, 77

vaccination, 76
Antiformin, 390
Antigen, 173, 175, 178

bacterial, 185
Antitoxin, 105, 164

botulism, 82, 124
. diphtheria, 102, 382, 384

pyocyaneus, 129

tetanus, 85
Antivenins, 380
Appendicitis blood count, 233

organisms in, 61
Arachnoidea, 334
Argas, 340

Arneth index, 227, 228
Ascaris, canis, 331

lumbricoides, 330
Ascitic fluid (cytodiagnosis in), 424
Aspergillus, concentricus, 142

flavus, 142

fumigatus, 142

nidulans, 143

pictor, 143

repens, 142

Auchmeromyia luteola, 357
Autopsy culture methods, 56
Azolitmin, 29

Babesia, 291

Bacillus, acidi lactici, 128, 157

acidophilus, 128

acnes, 421

aerogenes capsulat., 81, 87

Aertyrck, 123, 433

anthracis, 75

anthracis symptomat., 77

anthracoides, 77

bifidus, 128

Bacillus, botulinus, 82, 124
bulgaricus, 128, 157, 416
chauvoei, 77
cloacae, 128
coli, 127, 151, 153
coli anaerogenes, 154
coli, in water, 152
diphtherias, 102
dysenteriae, 125
enteritidis (Gartner), 124
enteritidis sporogenes, 87, 149
fecalis alkaligines, 119, 154
fusiformis, 387
icteroides, 119
influenzas, in
lactis asrogenes, no, 127
leprae, 97
mallei, 101
mycoides, 73, 74
of avian tuberculosis, 93, 94
of Bordet-Gengou, 113, 438
of bovine tuberculosis, 93
of chancroid, 113
of chicken cholera, 114
of Danysz virus, 117, 124
of Hofmann, 107
of hog cholera, 114, 433
of Koch-Weeks, in
of malignant oedema, 81, 82
of Morax, 112
of smegma, 90, 96
of timothy grass, 91
of trachoma (Muller), 109
of Zur Nedden, 113
paratyphosus (A. and B.), 123
perfringens, 88
pestis, 114

phlegmonis emphysematosae, 75
pneumonias (Friedlander) ,110,114
prodigiosus, 130
proteus, 124
pseudotuberculosis rodentium,

116

psittacosis, 119
pyocyaneus, 129
subtilis, 73
tetani, 84

INDEX

485

Bacillus, termo, 125, 395

tuberculosis, 92

tularense, 383

typhi-exanthematici, 438

typhosus, 119

violaceus, 129

vulgatus, 73

xerosis, 108

zopfii, no

Bacteria, identification of, 48
Bacteriaemia, 414
Bacteriuria, 401
Balantidium coli, 273
Banti's disease, 240
Bartonella, 440
Bed-bug, in Kala azar, 268
Belascaris, 331
Bence-Jones albumin, 458
Benedict sugar test, 460
Beriberi, 438
Besredka medium, 24
Bienstock group, no
Bile acids in urine, 466
Bile media, 31
Bile pigments, 408, 417, 466
Bilharziasis, 300

infection in, 303
Binucleata, 250

Blackwater fever, 292, 401, 439
Blastocystis hominis, 2 72
Blood, chemical examination of, 454
Blood coagulation rate, 215

color index of, 222

counting red cells, 203

counting white cells, 204

counting with microscopic field,
2.05

cultures of, 413

differential count (normal), 228

differential count (in haemacytom-
eter), 206

dried films, 208

fixation of, 210

fresh preparations, 206

haemoglobin in, 200

making preparations, 208

non-protein nitrogen in, 455

Blood, normal count, 222

occult, 216

red cells of, 222

specific gravity of, 215

spectroscopic test, 218, 394

staining of, 211

thick films of, 210

tubercle bacilli in, 415

tubes for collection, 18

urea in, 455

viscosity of, 214

white cells of, 224
Blood platelets, 230
Blood serum, coagulating apparatus,

12

preparation of, 30
Blood sugar, 454
Boas-Oppler bacillus, 416
Bodo, 272
Booker group, no

Bordet and Gengou bacillus, 113, 438
Bordet and Gengou phenomenon, 172
Bothriocephalus, 309
Bottle bacillus, 421
Botulism, 83
Bouillon, glycerine, 25

calcium carbonate, 25

egg, 25

Liebig's extract in, 23

nutrient, 20

standardizing reaction of, 22

sterilization of, 23

sugar, 24

sugar-free, 23
Bovine tuberculosis, 93
Bronfenbrenner's anaerobic culture, 36
Broth media, 20
Buccal secretions, 386
Burker's haemacytometer, 202

Calliphora vomitoria, 357
Calmette reaction in Tb., 95
Capillary pipettes, 17

standardizing of, 181
Capsule staining, 43
Carbol-fuchsin stain, 41
Carriers, in cholera, 133

486

INDEX

Carriers, in diphtheria, 102, 108, 384

in dysentery, 255

in typhoid fever, 122
Casts in urine, 399
Cellia, 373

Cells, in cytodiagnosis, 424
Centrifuge, 16
Cercomonas, 272
Cerebrospinal fluid, 425

puncture for, 425

Wassermann of, 173, 428
Cestoda, 294, 303

key to genera, 305
Charcot-Leyden crystals, 389, 405
Chinosol, 476

Chlorides in urine, 403, 466
Chlorinated lime, 476
Chlorosis, 235
Cholecystitis, 418
Cholera, 131

carriers in, 133

diagnosis, 135

in water, 155

media for, 34
Cholera red, 25, 135
Chironomidae, 363
Chlamydozoa, 293, 434
Cholesterinized antigen, 173
Chromatin stains, 212
Chromidia, 250, 258
Chromogens, 129
Chrysomyia macellaria, 357, 359
Chrysops, 353
Chyluria, 318, 396
Citellus, 351
Cladorchis watsoni, 298
Cladothrix, 136
Classification, animal kingdom, 244

arachnoidea, 334

bacilli, branching, 90

bacilli, gram negative, 109

bacilli, spore bearing, 73

bacteria, 51

cocci, 57

filarial worms, 320

flat worms, 294

fungi, 136

Classification, insects, 344

mosquitoes, 373

myiasis larvae, 361

protozoa, 249

round worms, 313

spirilla, 131
Cleaning fluid, 10
Clonorchis endemicus, 297

sinensis, 297
Coarctate pupa, 354
Cocci diaria, 282
Coccidium (see Eimeria and Isospora),

283

Cockroach, destruction of, 478
Coley's fluid, 130
Colloidal gold test, 426
Colon bacillus, 127, 153, 154

in water, 152
Colonies, isolation of, 50
Color index, 222
Colubrine snakes, 377, 379
Commensalism, 246
Common cold, 435
Complement, 165, 172, 183, 185

absorption of, 172

deviation of, 171
Complement fixation, bacterial, 185
Conjunctival infections, 381
Conorhinus, 348
Conradi-Drigalski medium, 34
Conradi-brilliant green medium, 34
Copper sulphate, 476
Corrosive sublimate, 476
Coryza, 435
Cover-glasses, 3

Cover-glass preparations, 38, 208
Crithidia, 269
Cryptococcus gilchristi, 139

linguae pilosae, 139
Ctenopsylla musculi, 350, 351
Culicinae, 374
Culture media, agar, 26

autolyzed tissue, 35

Besredka's, 24

bile media, 31

blood agar, 31

blood serum, 30

INDEX

487

Culture media, bouillon, 20

cholera media, 34

Dorsett's egg medium, 30

egg media, 30

faeces media, 33

gelatin, 28

gelatin agar (North), 28

Hiss' serum water, 25

litmus milk, 28

milk agar, 31

Noguchi media, 36

peptone solution, 25

Petroff's Tb. medium, 32

potato, 29

protozoal, 35

roll, 36

Russell's double sugar, 35

starch medium of Vedder, 27

sterilization of, 8

sugar bouillon, 24

titration of, 21
Cycloleppteron, 373
Cysticercus, 305
Cystitis, 401
Cytodiagnosis, 423
Cytorrhyctes luis, 293

scarlatinas, 293

vaccines, 293, 433

Dark ground illumination, 6
Davainea madagascariensis, 306, 309
Demodex folliculorum, 327
Deneke's spirillum, 131
Dengue, 439

Dermacentor andersoni, 342, 437
Dermatobia cyaniventris, 358, 359
Desk-microscopic, 12
Dhobies itch, 142, 145
Diazo reaction, 465
Dibothriocephalus latus, 309
Dicroccelium lanceatum, 297
Dieudonne's cholera medium, 34
Differential leukocyte count, 228,

229
Diphtheria, 102

carriers, 102

diagnosis of, 106

Diphtheria, diphtheria-like bacilli, 107

media for growing, 30

Neisser's stain, 42

Schick reaction, 106

toxin of, 104
Diphtheroids, 108
Diplococcus, crassus, 58

intracellular, meningitidis, 68

lanceolat., 64

Diplognoporus grandis, 310
Diptera, 351

Dipylidium caninum, 309
Disinfecting solution, 13, 476
Disinfectants, 472

solutions for laboratory, 13
Distomiasis, 297
Dorset's egg medium, 30
Double boiler, 12, 19
Dracunculus, 315
Dum dum fever, 267
Dunham's solution, 25
Duodenal fluid, 417, 472

examination of 472

tests for ferments in, 472
Dysentery, amoebae in, 252

bacilli, 125

bacilli in faeces, 409

Ear affections, 385

Eberth group, 119

Echinococcus cysts, 310

Echinorhynchus gigas, 332

Echinostoma, 299

Edestin test, 471

Egg broth, 25

Ehrlich, blood film method, 208

granule staining, 213, 227

tri-acid stain, 211
Eimeria stiedae, 283
Emery's test, 179
Ekiri, 127

Endodermophyton, 141
Endo medium, 33
Endomyces albicans, 139
Endothelial cell in cytodiagnosis,

424
Entamceba, buccalis, 258

INDEX

Entamoeba, coli, 252

gingivalis, 258

histolytica, 252

tetragena, 252
Eosinophiles, 227, 231
Eosinophilia, 231
Epidermophyton, 142
Esbach test, 458
Escherich group, 119
Esmarch roll cultures, 50
Espundia, 270, 422
Eusol, 477

Eustrongylus gigas, 324
Exudates, 423
Eye-piece (see Ocular), 2
Eye-strain, 4
Eye infections, 381

Faeces, 405

amoebae in, 408

bile in, 408

culturing, 33, 409

diet for examination of, 406

fats in, 407

fermentation test, 408

pancreatic test, 410

plating media, 33

soaps in, 407
Fasciola gigantea, 300

hepatica, 297
Fascioletta ilocana, 299
Fasciolopsis buski, 298
Fat in faeces, 407
Fauces, 386
Favus, 141

Fehling sugar test, 459
Fermentation tubes, 1 1
Ferments in duodenal fluid, 417, 472
Films (blood), 208, 210
Filter pump, 16
Filterable viruses, 433
Filaria, bancrofti, 317

demarquayi, 319

embryos, key to, 319

loa, 317

medinensis, 315

ozzardi, 319

Filaria, perstans, 318

philippinensis, 318, 319

powelli, 319

volvulus, 319
Fixation, blood films, 210

tissues, 443
Flagella staining, 44
Flagellata, 259
Flat worms, 294
Fleas, 348

key, 349

Flugge's droplet infection, 93, 118
Flukes, 294

of blood, 300

of intestines, 298

of liver, 297

of lungs, 299
Focus, microscopical, 3
Fontana spirochaete stain, 47
Foot and mouth disease, 436
Formalin, 443, 474
Foulis' cells, 424
Friedlander group, no, 114
Frozen sections, 450
Fungi, Achorion, 141

Ascomycetes, 138

Aspergillus, 142

classification of, 136

Cryptococcus, 139

cultivation of, 147

diagnosis of, 147

Discomyces bovis, 143

Discomyces madurae, 144

Hyphomycetes, 143

Inperfecti, 143

Madurella mycetomi, 145

Malassezia furfur, 145

Microsporoides, 145

Microsporum audouini, 141

Monilia, 145 ,

Mucor, 138

Penicillium, 143

Rhizopus, 138

Saccharomycetes, 139

Sterigmatocystis, 143

Trichophyton, 140

Trichosporum giganteum, 146

INDEX

489

Gall stones, 410
Gametes, 285
Gartner group, 123
Gas bacillus, 81, 87
Gas gangrene, 81
Gas production, 54
Gastric contents, 416, 469

chemical examination of, 469
Gastric ulcer streptococci, 61
Gastrodiscus hominis, 298
Gelatin, 28

liquefaction of, 54
General paralysis (spinal fluid in), 174,

426

Gentian violet stains, 39
Giemsa's stain, 45, 213
Glanders, 101

Glassware, cleaning of, 9, 427
Globulin tests, 427
Glossina palpalis, 355
Gluzinski test, 469
Gnathostoma siamense, 314, 422
Gonococcus, 67
Gonorrhoea, 67
Goundou, 440
Grabhamia, 376
Gram method, 39

negative bacteria, 40

positive bacteria, 40

solution, 39

Granular degeneration (red cells), 223
Granules (white cells), 226
Guinea worm, 315

Haemacytometer, 202
Haemadipsa ceylonica, 333
Hsematopota, 353
Haematoxylin stain, 46, 213, 449
Haematuria, 401
Haemin crystals, 217
Haemoglobin estimation, 200
Haemoglobinometers, Miescher's, 200

Sahli's, 200

Tallquist, 201

Haemoglobinophilic bacteria, in, 112
Haemoglobinuria, 216, 401
Haemolysin production, 176

Haemolysis tests, 176
Haemosporidia, 283
Haffkine, cholera vaccine, 134

plague prophylactic, 134
Halzoun, 297
Hanging drop, n
Hayem's blood diluent, 203
Hemokonia, 230
Heredity, 247
Herpetomonas, 269
Heterogenesis, 248
Heterophyes heterophyes, 299
Hirudo, medicinalis, 332

nilotica, 332
Hiss' serum-water, 25
Histoplasma, 271
Hodgkin's disease, 239
Hog cholera virus, 433
Homology, 248
Hook worms, 325
Hoplopsyllus, 349
Hosts, 247
Hydatid disease, 310
Hydrocele agar, 31
Hydrogen dioxide, 476
Hymenolepis, nana, 307

diminuta, 309
Hyphomycetes, 143
Hypoderma diana, 358

Illumination, dark ground, 6
Immersion objectives, 3
Immune sera, antimicrobic, 164

antitoxic, 163

diphtheria, 105

in diagnosis, 166

preparation, 166

tetanus, 85
Immunity, active, 163

natural, 161

passive, 163

Inactivation of serum, 165
Incubators, body temperature, 15

electrical, 15

petroleum lamp, 15

room temperature, 15
Indican in urine, 465

4QO

INDEX

Indol, test for, 25
Influenza, in
Infusoria, 273

Inoculation animals (tuberculosis) ,
48, 91

animals (plague), 116

of media, 49
Insecticides, 477
Inspissators, 12
Insecta, 344
Intestinal amoebae, 252

flagellates, 271

myiases, 360
lodophilia, 214
Isospora bigemina, 283
Itch mite, 337
Ixodidae, 338

Japanese river fever, 336, 442
Joints, gonococcus in, 68

Kaiserling solution, 452

Kala azar, 267

Kedani mite, 336, 442

Key to branching, curving bacilli, 90

to cocci, 57

to filarial embryos, 320

to fleas, 349

to Gram-negative bacilli, 109

larvae in myiases, 361

to spirilla, 131

to spore-bearing bacilli, 73
Kidney diseases, table, 404
Koch's postulates, 55
Kundrats lymphosarcoma, 240

Laboratory desks, 13
Lactic-acid bacteria, 128, 156
Lactophenol, 451
Lamblia intestinalis, 273
Lamp, primus, 18
Lange's colloidal gold test, 426
Large mononuclear increase, 234
Larvae, fly, 361

key to dipterous, 361

mosquito, 368

mounting, 451

Leeches, 332
Leishmania, 267

donovani, 267

infantum, 268

media for, 35

tropica, 269
Leishmaniases, 267
Leprosy, 97

cultural questions, 98

diagnosis of 83, 100

in rats, 99
Leptomonas, 269
Leptothrix, 136
Leukaemia, 238

lymphatic, 239

splenomyelogenous, 238
Leukocytosis, 232
Leukopenia, 231
Levaditi stain, 447
Leydenia gemmipara, 249
Lice, 345, 478

destruction of, 478
Light in microscopical work, 5
Linguatula rhinaria, 343
Liquefaction of gelatine, 54
Litmus, 29
Liver abscess, 420
Loeffler serum, 30
Loemopsylla cheopis, 350
Luetin, 262
Lumbar puncture, 425
Lymphocytes, large, 225

small, 225

Lymphocytosis, 234
Lymphosarcoma, 239
Lyon's blood tube, 18

Madura foot, 144
Macrogamete, 285
Magnifying power, 199

of oculars, 2
Malaria, 284

cultivation, 289

diagnosis of, 286

differential tables, 290

index, 286

life cycle, 284

INDEX

491

Malaria, life history, 284

Romano wsky stain in, 291
Mallein, 102

Mallory's amoeba stain, 46
Malta fever, 71
Mansonia, 376
Marchi method, 449
Mast cells, 227
Measles, 436
Megarhininae, 373
Meat poisoning, 83, 123

group of bacteria, 123

toxin of, 83
Malignant pustule, 75
Mechanical stage, I
Media (see culture media), 19
Megaloblast, 224
Megakaryocytes, 230
Melaniferous leukocytes, 235
Meningococcus, 68
Metorchis truncatus, 298
Mett test for pepsin, 471
Micrococcus, 62

catarrhalis, 70

cinereus, 58

melitensis, 71

pharyngis siccus, 58

rheumaticus, 61, 435

tetragenus, 62
Microgametocyte, 285
Micrometer disk, 197

standardization of, 198

screw, i

Micro metry, 197
Microscope, i

care of, 2

dissecting, i
Microscopical sections (see tissue), 443

quick diagnostic method, 446
Milk, bacteriological examination of,

155

B. bulgaricus in, 157

lactic-acid bacteria in, 157

leukocytes in, 157

pasteurization of, 158
Minimal lethal dose, 105
Mites, 335

Mollusc hosts in schistosomiasis, 303
Monilia albicans, 145, 441

Candida, 146

in sprue, 442

Mononuclear, leukocytes, 225
Mosquitoes, anatomy of, 365

classification of, 372

dissection of, 370

larvae of, 368

ova of, 367

pupae of, 369

transmitters of malaria, 373
Motility, 53

Brownian, 51

current, 51-53
Moulds (see Fungi), 136
Mounting parasites, 450
Much's granules, 41
Mucidus, 376
Mumps, 437
Mus norvegicus, 351
Musca domestica, 353
Muscidae, 353
Mutualism, 245
Myeloblasts, 230
Myelocytes, 229
Myiases, 359

key to larvae in, 361
Myzomyia, 373
Myzorhynchus, 373

Nasal infections, diphtheria in, 384

leprosy in, 384
Nastin in leprosy, 99
Necator americanus, 325
Negri bodies, 430
Neisser's stain, 42
Nematocera, 352
Nematoda, 313
Neosporidia, 282
Nervous tissue, 449
Nissl method, 449
Nitrogen determination, 455-462
Nocardia, 147
Noguchi test, 174

media for treponemata, 36

492

INDEX

Nomenclature, in animal parasitology,

246

law of priority in, 246
Nonprotein nitrogen, 455
Normal solutions, 452
Normoblasts, 223
North's gelatin agar, 28
Novy MacNeal (N.N.N.) medium, 35
Nucleo-protein test, 458
Numerical aperture, 4
Nylander sugar test, 461
Nyssorhynchus, 373

Objectives, 2
Occult blood, 216
Ocular infections, 381

animal parasites in, 381

bacilli in, 382

gonococcus in, 382

M. catarrhalis- in, 383

pneumococcus in, 382
Oculars, 2

CEsophagostoma brumpti, 325
CEstridae, 358
Oidium, 145

Onchocerca volvulus, 319
Opisthorchis, felineus, 298

noverca, 298

sinensis, 297
Opsonic power, 1 86

apparatus in, 188

determination of, 188
Ornithodoros, 340
Oroya fever, 440
Orthorrhapha, 351
Otitis, 385
Ova in faeces, 411
Oxyuris vermicularis, 331

Pancreatic tests, 410, 472
Pandy globulin test, 428
Pangonia, 353
Panoptic staining, 46, 448
Paragonirhus westermani, 299
Paraplasma flavigenum, 292, 442
Parasitism, 243
Parthenogenesis, 248

Pasteur elloses, 114
Pasteurized milk, 158
Pasteur treatment, 429
Pathological sections, 443
Pebrine, 282
Pediculicides, 478
Pediculoides ventricosus, 337
Pediculus capitis, 345

vestimenti, 345
Pellagra, 440

Penicillium crustaceum, 142
Pentastomida, 342
Pepsin tests, 471
Petri dishes, 9, 48
PetrofFs T b. medium, 32
Pfeiffer's phenomenon, 134
Phagocytosis, 186
Pharyngeal secretions, 386
Phenol coefficient, 473
Phenolphthalin test, 217
Phenolsulphonephthalein test, 467
Phenylhydrazin test, 459
Phlebotomus, 364
Phthirius pubis, 346
Physaloptera, 325
Physiological normals, 480
Piedra, 146
Pinta, 143
Pipettes, bacteriological, 18

capillary bulb, 17
Piroplasmata, 291
Pirquet, von, reaction in T b., 96
Plague, 114

diagnosis of, 116

flea in, 117, 349

pneumonia, 118

prophylaxis, 118
Plasmodium vivax, 287

falciparum, 288

malarias, 288
Platinum wire, 14

loops, 14

Pleural fluids (cytodiagnosis), 423
Pneumococcus, 64
Poikilocytes, 223
Poliomyelitis, 436
Polymorphonuclear leukocytes, 227

INDEX

493

Porocephalus constrictus, 343
Precipitins, 166-170
Protista, 248
Protozoa, 250

culture of, 35

discussion of, 250

staining of, 45
Prowazekia, 272
Pseudoleukaemia, 239
Psychodidae, 364
Pulex, cheopis, 350

irritans, 350
Pulicidae, 348
Pupipara, 352
Pus, cultures from, 419

in urine, 401

tetanus in, 86
Pyorrhoea, amoebae in, 258
Pyosis, 63
Pyretophorus, 373

Rabies, 429

preservation of dog in, 431

Rats, 351

Rat-bite disease, 441

Reaction of media, 21, 51
standardization of, 21

Reagine of syphilis, 173

Red blood-cells, counting of, 203
normal, 222
nucleated red cells, 223
polychromatophilia, 223
punctate basophilia, 223

Relapsing fever, 260

Renal efficiency determinations, 402

Rhabditis pellio, 314

Rheumatism (acute), 435

Rhinosporidium, 292

Rhizoglyphus parasiticus, 337

Rhizopoda, 251

Rhizopus, 138

Rhynchota, 346

Rice cooker, 12, 19

Ring- worms, 140

Rocky Mountain spotted fever, 437

Roetheln, 437

Romano wsky stains, 212 .

Room temperature incubators, 15

Ross thick film, 210

Round worms, 313

Row's haemoglobin medium, 36

Russell's double sugar medium, 35

Sabouraud's medium for moulds, 148
Saccharomyces, anginosse, 139

blanchardi, 139

cerevisiae, 139
Salt retention, 403
Sarcina lutea, 62
Sarcophaga carnaria, 357-361
Sarcopsylla penetrans, 351
Sarcoptes scabiei, 337
Sarcosporidia, 292
Scarlet fever, 61, 437
Schick reaction, 106
Schilling-Torgau differential count,

229
Schistosomum haematobium, 300

japonicum, 302

mansoni, 301
Schizotrypanum, 266
Screw worm, 359

Sections, making and staining, 443
Sensitization, 165
Sensitized vaccines, 192
Septicaemia, 414

Serum (see immune serum), 163-165
Sewage, in water, 149
Sheep cells, 178
Shiga's bacillus, 125
Simulidae, 363
Siphonaptera, 348
Siphunculata, 345
Skin infections, 42 1

itch mite, 337, 422

leprosy in, 421

pus cocci in, 421

sarcopsylla in, 422
Sleeping sickness, 264
Slides, cleaning, 10

concave, n
Smallpox, 432
Snakes, 377
Sparganum, mansoni, 312

494

INDEX

Sparganum, prolifer, 312
Spectroscope, 218, 393
Spinal fluid, 173, 425
Spirillum cholerae asiaticae, 131

metschnikovi, 131

of Finkler-Prior, 131

tyrogenum, 131
Spirochaeta, 259

duttoni, 260

recurrentis, 260

refringens, 261

vincenti, 261, 386
Spirochaete staining, 47
Spiroschaudinnia, 260
Splenic anaemia, 241
Splenomegaly, 240
Spores, spore-bearing bacilli, 73

staining, 45
Sporotrichosis, 146
Sporotrichum beurmanni, 146
Sporozoa, 282
Sprue, 441
Sputum, 389

albumin test in, 392

amoebae in, 392

antiformin for, 390

centrifugalization for T. B., 390

culturing, 32, 391

fixing smears, 391

Paragonimus eggs in, 392

Petroff 's T b. culturing, 32

plague pneumonia, 392
Stage, warm, 5
Staining methods, 38
Stains, acid fast, 41

agar jelly, 45

Archibald's, 42

Balch's, 212

capsule, 43

carbol fuchsin, 39

carmine for worms, 45 1

flagella, 44

Fontana spirochaete, 47

for Negri bodies, 431

Giemsa's, 213

Gram's method, 39

haematoxylin, 46, 213, 449

Stains, Herman's, 41

iron haematoxylin, 46, 447

Leishman's, 212

Levaditi's, 447

Loffler's methylene blue, 39

Mallory's, 46, 47, 449

Neisser's, 42, 107

Nicolle's, 447

panoptic, 46, 448

Pappenheim's, 42

Ponder's diphtheria, 43

protozoal, 45

Romano wsky, 212

Rosenbusch's, 46

Smith's formol fuchsin, 41

spore, 45

tri-acid, 211

Van Giesen's, 447

Wright's, 212
Staphylococcus, 63

epiderrriidis albus, 63, 421

pyogenes albus, 63

pyogenes aureus, 63
Stegomyia, 374
Sterigmatocystis, 143
Sterilization, Arnold, 7

autoclave, 8

glass ware, 9

hot air, 6

pathogenic bacteria, 9
Stomach contents, 416, 469

Boas-Oppler bacillus, 416

cancer cells in, 416
Stomoxys, 355
Stool examination, 405
Streptococcus, 58

anaerobic, 414

capsulatus, 65

coli gracilis, 57

fecalis, 57

haemolyticus, 60

lacticus, 156

mucosus, 65

pyogenes, 60

viridans, 60
Strep tothrix, 136
Strong, cholera prophylactic, 134

INDEX

495

Strong, plague vaccine, 118
Strongylidae, 324
Strongyloides stercoralis, 314
Strongylus apri, 324
Sugar in blood, 454

in urine, 459

media, 24, 153
Sulphur disinfection, 475
Swabs, 9

Syphilis, fixation of complement in
diagnosis, 173

Giemsa's stain in, 261

precipitate tests in, 185

smears in, 262

Tabanus, 352
Tables, insects, 344

mosquitoes, 373

of arachnoids, 334

of filarial worms, 320

of flat worms, 294

of fungi, 136

of myiasis larvae, 361

of parasitic animals, 245

of protozoa, 249

of round worms, 313

pressure and temperature, 9

urinary findings, 404
Taenia, africana, 307

echinococcus, 310

saginata, 306

solium, 307
Taeniorhynchus, 376
Tape worms, adult, 303

somatic or larval, 310
Telosporidea, 282
Terminology, 247
Test-tubes, 9
Tetanus, 84

antitoxin, 85

diagnosis of, 81, 86

toxin, 85

Tetragena nucleus, 253
Tetramitus mesnili, 272
Thalman's gonococcus medium, 32
Theobaldia, 376
Thick film blood smears, 210

Thionin, 42, 211
Thorn-headed worm, 332
Throat examination, 386
Thrush, 139, 145, 386
Tick fever, 260, 340
Ticks, 338

classification of, 334

life history, 339
Tinea, 140

imbrica^a, 141, 142

versicolor, 145
Tissue, acetone method, 445

chloroform method, 445

dehydration of, 444

fixation of, 443

imbedding, 444

nervous, 449

preparation of for sections, 443
Titration of media, 21
Toisson's solution, 203
Tongue worms, 342
Toxascaris, 331
Toxic split proteids, 194
Toxin, 164

diphtheria, 105

tetanus, 85

Toxone of diphtheria, 105
Trachoma, 112, 437

and Koch- Weeks bacilli, 112
Transfusion blood tests, 216
Transitional leukocyte, 226
Transudates, 423
Trematoda, 294
Treponema, 261

culture media for, 36

pallidum, 261

pertenue, 263
Trichinella spiralis, 321
Trichinosis, 321
Trichocephalus trichiurus, 321
Trichomonas intestinalis, 272

vaginalis, 272, 395
Trichophyton mentagrophytes, 141

Sabouraudi, 140

tonsurans, 140

Trichostrongylus instabilis, 324 .
Trichuris, 321

496

INDEX

Triodontophorus deminutus, 324
Trombidiidae, 335
Trombidium holosericeum, 335
Trypanocides, 264
Trypanosoma, brucei, 266

equinum, 266

equiperdum, 266

evansi, 266

gambiense, 264

lewisi, 266

rhodesiense, 264
Trypanosomiasis, 264
Tsetse flies, 265, 355
Tuberculin, 95
Tuberculosis, bacilli of, 92

atrium of infection, 93

avian and fish, 94

bovine and human, 93

British commission report, 93

Calmette eye reaction, 95

diagnosis of, 95

in guinea-pigs, 91

intradermic test, 96

Much's granules, 41

secondary infections, 94

staining methods, 41

von Pirquet skin reaction, 96
Tuberculins, 95
Tubes, fermentation, 8

test-tubes for agglutination, 169

Wright's U-tube, 17
Turck, irritation cells, 229

ruling on haemacytometer, 202
Typhoid, agglutination in, 121

and water supply, 123, 155

blood cultures, 121, 412

carriers, 122

dead cultures, 169

gall stones in, 122

in water, 155

Mandelbaum's thread culture, 1 70

relapses in, 121

serum for, 121

statistics, 123

vaccination in, 122
Typhus fever, 438
Tyroglyphus longior, 337

Ultra microscope, 6
Urea, 393

in blood, 455

in urine, 463

index of excretion, 402
Urease tests, 455, 464
Uric acid tests, 466
Urine, 393

casts in, 399

chemical examination, 456

chylous, 396

culturing of, 395

epithelium in, 399

moulds in, 395

reaction of, 462

schistosomum eggs in, 396

sediment in, 396

smegma bacillus in, 395

starch grains and fibres in, 400

sugar in, 459
Urobilin, 466
Urotropin and urine, 467
Urticarial fever, 302
Uta, 422

Vaccines, 189

doses of, 191

preparation of, 189

sensitized, 192

standardizing of, 191

typhoid and paratyphoid, 124
Vaccinia, 432
Vacuum bottle, 80
Varicella, 438

Vedder's starch medium, 27
Verruga peruana, 442
Vincent's angina, 386
Viperine snakes, 379
Vitamines, 439
Voges-Proskauer, 25

Warm stages, 5
Wassermann test, 177

Emery, 179

Fleming, 184

Noguchi, 174

statistics, 184

INDEX

497

Water, bacteriological examination,
149

cholera spirillum in, 155

colon bacillus in, 152

disinfecting, 477

qualitative bacteriological exami-
nation, 151

quantitative bacteriological ex-
amination, 150

typhoid bacillus in, 155
Weigert method, 449
Whip worms, 321
White blood-cells, 224

counting of, 204

normal count, 228, 229
Whooping cough, 438
Widal tests, 168
Wing venation, 352-354

Wollf and Junghan's test, 470

Woolsorter's disease, 75

Working distance, 3

Wright's blood stain, 212

method for anaerobes, 80
method for standardizing
cines, 190

Xenopsylla cheopis, 350
Xerosis bacillus, 108

Yaws, 263
Yersin's serum, 118
Yellow fever, 442

Zettnow's flagella stain, 44
Zur Nedden's bacillus, 113

p

115
I 10

10*

100

xSo

Equivalent Fahrenheit and
Centigrade tables for the temper-
atures in common use in labora-
tories:

i. Those for sterilization of
dressings and media. Also f for
certain disinfection of spore-bear-
ing bacterial contamination
(autoclave temperatures).

P. C.

2. Those for pasteurization
and sterilization of bacterial vac-
cines. Also for paraffin bath
(pasteurizing temperatures).

1*0

57

V04-

1*

~s^

f

—

75

x

^

—

1

—

GQ.S

to

£

—

5

\ —

ji-

p-i

4-

—

•*0-

5

_

t

• — •

a

_

^

H

i

—

V

-

r\

~

32.

3. Those for growing impor-
tant pathogenic organisms (body
temperatures).

4. Those for culturing gelatin
(melting point, 25°C.) as in water
work (room temperatures).

5. Those employed in preserv-
ing biological products and post-
mortem material; also in centri-
fuging experiments to separate
complement and amboceptor
(freezing temperatures).

160

50 =

320

10 =

10

AMERICAN STERILIZER co,

ERJE.F*.

UNITS IN COMMON USE IN LABORATORIES

Cubic Meter. — Unit of space for the number of organisms in air. It
contains 1000 liters. It is equal to 1.308 cubic yards or 35.316
cubic feet. One thousand cubic feet, the unit of space in disinfec-
tion, is equal to 28.3+ cubic meters. One cubic decimeter is one
liter and equals 0.908 quarts dry measure or 1.0567 quarts liquid
measure.

Liter. — Unit of space for normal volumetric solutions. It contains
1000 cubic centimeters. It is equal to 1.0567 quarts or 33.84-
ounces. A liter of distilled water weighs i kilogram. One U. S.
gallon is equal to 3785 c.c. and one imperial gallon to 4543 c.c.
One fluid ounce equals 29.57 c.c.

Cubic Centimeter. — Unit of space for organisms in water, milk, vac-
cines, etc., i c.c. = 0.27 fl. dr. There are, approximately, 20 drops
in i c.c. of water, provided the capillary pipette has a bore of about
i mm. and is held horizontally. A finely drawn capillary pipette,
held vertically, will deliver about 50 drops from i c.c.

Cubic Millimeter. — Unit of space for blood-cells. There are 1000
cubic millimeters in i cubic centimeter and i million cubic milli-
meters in i liter. In water analysis, as there are i million milli-
grams in one liter, parts in the million and milligrams per liter
are the same.

i Meter = 39. 3 7 inches.

i Centimeter = 0.393 7 inch. Approximately, 2/5 inch.

i Millimeter = 0.0393 inch. Approximately, 1/25 inch.

i Inch = 2 5. 4 mm.

i Yard = 0.9 144 m.

i Kilogram = 2. 2+ pounds av.

i Centigram = 0.154 grain.

i Milligram = 0.01 54 grain. Approximately, 1/64 grain.

A pound avoirdupois is equal to 453.59 gm.

i Oz. avoirdupois is equal to 28.35 gm.

One hundred cubic centimeters of a saturated solution contains:

Water Alcohol

Methylene blue, 6.68 0.66 gram.

Gentian violet, 1.75 4.42 grams.

Basic fuchsin, 0.66 2.92 grams.

Key to Table on opposite page.

— = negative + = positive, O = no change, A = acid, Alk. = alkaline, G — 6««,
Fl = fluorescence, Pep = peptonization, i= litmus Russell's medium not reduced;
2 = reduced, 3 = action variable. Proteus gives a peculiar dark, heavy oil-li1
fluorescence in neutral red glucose bouillon.

Remarks

Found in faeces and sewage-contami-
nated water. Differs from B. typhos.
by marked alkali production.

Blood cultures first week — agglutina-
tion afterward.

Nonacid strain, highly toxic.

Acid mannite strain, moderate toxicity.

Much like Flexner strain. No acid in
maltose.

Found in summer diarrhoea of children.

Little gas. No fluorescence n. red.
Litmus milk acid in third day. i.

Much gas. Marked reduction n. red
with yellow fluorescence. Litmus
milk alkaline third day. 2.

B. choleras suis, B. icteroides, B. of
Danysz virus and B. paratyphoid B.
closely related (Gaertner group). 2.

There is also a B. coli anaerogenes which
is like B. coli but does not form gas.

Very nearly related to Friedlander's
bacillus as well as to B. coli. 3.

Differs from B. coli in liquefactio* of
gelatin and shows slow production
of gas in lactose.

Three types — Proteus vulgaris rapid
gelatin liq.; P. mirabilis, slow gelatin
fiq.; P. zenkeri, no gelatin liq. Spread-
ing growths characteristic, a.

janT^sojci-saaoA

1

1

1

1

1

1

1

1

I

1

+

+

•

I°PUI

I

1

1

1

•H

+

1

1

1

+

•H

+

1

|j 1

0

<

3

3

3

3

a

1

<

<

<

M

jj (0 g

W

O

<

<2

<

<J

O

o

O

O

O

O

O

**<

.ssassio

0

0

0

o

0

0

0

6

o

E

o

O

O

O

E

o

—*»-8

o

O

0

o

0

0

o

0

0

0

0

o

0

a^nnrep^

0

4

0

<

-i

o

o

o

o

o

o

o

<J

o

aso^oBi

0

O

o

o

O

0

0

0

0

o

o

o

0

,So,™

0

<

0

<

0

0

o

o

o

o

o

o

0

asoonio

0

<

<

4

<

0

o

0

o

o

o

o

o

a 1*

J«

!

3

3

3

1

3

,x

1

«

<

1

1

Til '

€

«

3

M

M

0

<

9

£

«

<

<

3

a «.>.

to oJ
wtQ

3

<

«

<

<

0

<

«

«

«

«

0

o

^uoi}tJin8eoD) snipi

1

i

1

\

1

1

1

1

1

+

+

+

+

aut^po

1

i

1

1

1

1

1

1 1

1

1

1

+

±

Alipioft

+

+

1

1

1

+

+

1 +

+

+

1

+

+

sajnsdBO

1

i

1

1

1

1

1

i 1

1

1

+

1

1

1.1
11

«ll

jo-a

faecalis alkaligines.

typhosus.

dysenteriae (Shiga-
Kruse).

dysenterias (Flex-
ner-Strong).

dysenteriae "Y."

Morgan No. I.

paratyphosus A.

j

I

1

I

enteritidis.
(Gaertner.)

8

. lactis aerogenes.

t

1

3

ui

e

pq

H

pq

pq
n

/*-

£3

pq

1

pq

^1

«

pq
o\

m

o

pq

M

m

pq

M